4-1BB binding molecules

ABSTRACT

The present disclosure provides isolated binding molecules that bind to human 4-1BB, nucleic acid molecules encoding an amino acid sequence of the binding molecules, vectors comprising the nucleic acid molecules, host cells containing the vectors, methods of making the binding molecules, pharmaceutical compositions containing the binding molecules, and methods of using the binding molecules or compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of non-provisional application Ser. No.13/228,532, filed on Sep. 9, 2011, now U.S. Pat. No. 8,337,850.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled“PC33845AUSSequenceListing_ST25.txt” created on Sep. 9, 2011 and havinga size of 77 KB. The sequence listing contained in the .txt file is partof the specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present disclosure relates to antibodies, and particularlyantibodies that bind to human 4-1BB.

BACKGROUND

4-1BB (also referred to as CD137, TNFRSF9, etc) is a transmembraneprotein of the Tumor Necrosis Factor receptor superfamily (TNFRS).Current understanding of 4-1BB indicates that expression is generallyactivation dependent and is present in a broad subset of immune cellsincluding activated NK and NKT cells, regulatory T cells, dendriticcells (DC), stimulated mast cells, differentiating myeloid cells,monocytes, neutrophils, and eosinophils (Wang, 2009, ImmunologicalReviews 229: 192-215). 4-1BB expression has also been demonstrated ontumor vasculature (Broll, 2001, Amer. J Clin. Pathol. 115(4):543-549;Seaman, 2007, Cancer Cell 11: 539-554) and at sites of inflamed oratherosclerotic endothelium (Drenkard, 2007 FASEB J. 21: 456-463;Olofsson, 2008, Circulation 117: 1292-1301). The ligand that stimulates4-1BB, i.e., 4-1BB Ligand (4-1BBL), is expressed on activatedantigen-presenting cells (APCs), myeloid progenitor cells, andhematopoietic stem cells.

Human 4-1BB is a 255 amino acid protein (Accession No. NM_(—)001561;NP_(—)001552). The complete human 4-1BB amino acid sequence is providedin SEQ ID NO:68. The protein comprises a signal sequence (amino acidresidues 1-17), followed by an extracellular domain (169 amino acids), atransmembrane region (27 amino acids), and an intracellular domain (42amino acids) (Cheuk A T C et al. 2004 Cancer Gene Therapy 11: 215-226).The receptor is expressed on the cell surface in monomer and dimer formsand likely trimerizes with 4-1BB ligand to signal.

Numerous studies of murine and human T cells indicate that 4-1BBpromotes enhanced cellular proliferation, survival, and cytokineproduction (Croft, 2009, Nat Rev Immunol 9:271-285). Studies haveindicated that some 4-1BB agonist mAbs increase costimulatory moleculeexpression and markedly enhance cytolytic T lymphocyte responses,resulting in anti-tumor efficacy in various models. 4-1BB agonist mAbshave demonstrated efficacy in prophylactic and therapeutic settings.Further, 4-1BB monotherapy and combination therapy tumor models haveestablished durable anti-tumor protective T cell memory responses(Lynch, 2008, Immunol Rev. 22: 277-286). 4-1BB agonists also have beenshown to inhibit autoimmune reactions in a variety of art-recognizedautoimmunity models (Vinay, 2006, J Mol Med 84:726-736). This dualactivity of 4-1BB offers the potential to provide anti-tumor activitywhile dampening autoimmune side effects that can be associated withimmunotherapy approaches that break immune tolerance.

There is a long-felt unmet need for antibodies that bind human 4-1BB,increase a 4-1BB-mediated response, and thereby provide a potentialtherapeutic for treatment of various diseases and conditions, includingcancer.

SUMMARY

It is an object of the disclosure to provide an isolated bindingmolecule that binds to human 4-1BB, such as an antibody or a bindingfragment thereof, or derivative thereof. It is another object of thedisclosure to provide a composition comprising a binding molecule thatbinds to 4-1BB. It is also an object of the present disclosure toprovide methods for treating a disease and/or condition associated withor mediated by 4-1BB signaling by using one or more binding molecules ofthe disclosure. These and other objects of the disclosure are more fullydescribed herein.

In some aspects, the disclosure provides isolated antibodies that bindto human 4-1BB.

In one particular aspect, the isolated antibody binds human 4-1BB at anepitope comprising amino acid residues 115-156 of SEQ ID NO: 68. In someparticular embodiments, the antibody comprises the H-CDR1 amino acidsequence of SEQ ID NO: 29, H-CDR2 amino acid sequence of SEQ ID NO: 30,and H-CDR3 amino acid sequence of SEQ ID NO: 31. In other particularembodiments, the antibody comprises the L-CDR1 amino acid sequence ofSEQ ID NO: 34, L-CDR2 amino acid sequence of SEQ ID NO: 35, and L-CDR3amino acid sequence of SEQ ID NO: 36.

In another particular aspect, the isolated antibody binds human 4-1BBwith a K_(D) of 600 nM or less, 100 nM or less, 50 nM or less, 10 nM orless, 5 nM or less, or 1 nM or less, for the human 4-1BB extracellulardomain as measured with the BIACore assay described in this disclosure.

In another particular aspect, the isolated antibody comprises: (a) anH-CDR1 as set forth in SEQ ID NO:1, SEQ ID NO:15, or SEQ ID NO:29; (b)an H-CDR2 as set forth in SEQ ID NO:2, SEQ ID NO:16, or SEQ ID NO:30;and (c) an H-CDR3 as set forth in SEQ ID NO:3, SEQ ID NO: 17, or SEQ IDNO:31.

In another particular aspect, the isolated antibody comprises: (a) anL-CDR1 as set forth in SEQ ID NO:6, SEQ ID NO: 20, or SEQ ID NO:34; (b)an L-CDR2 as set forth in SEQ ID NO:7, SEQ ID NO:21, or SEQ ID NO:35;and (c) an L-CDR3 as set forth in SEQ ID NO:8, SEQ ID NO:22, SEQ IDNO:36, or SEQ ID NO: 55.

In a further aspect, the isolated antibody comprises: (a) an H-CDR1 asset forth in SEQ ID NO:1, SEQ ID NO:15, or SEQ ID NO:29; (b) an H-CDR2as set forth in SEQ ID NO:2, SEQ ID NO:16, or SEQ ID NO:30; and (c) anH-CDR3 as set forth in SEQ ID NO:3, SEQ ID NO: 17, or SEQ ID NO:31; andfurther comprises: (d) an L-CDR1 as set forth in SEQ ID NO:6, SEQ ID NO:20, or SEQ ID NO:34; (e) an L-CDR2 as set forth in SEQ ID NO:7, SEQ IDNO:21, or SEQ ID NO:35; and (f) an L-CDR3 as set forth in SEQ ID NO:8,SEQ ID NO:22, SEQ ID NO:36, or SEQ ID NO: 55.

In some further particular aspects, the isolated antibody, is selectedfrom the group consisting of:

(a) an antibody or antigen-binding portion thereof, comprising: anH-CDR1 as set forth in SEQ ID NO:1, an H-CDR2 as set forth in SEQ IDNO:2, and an H-CDR3 as set forth in SEQ ID NO:3;

(b) an antibody or antigen-binding portion thereof, comprising an H-CDR1as set forth in SEQ ID NO:15, an H-CDR2 as set forth in SEQ ID NO:16,and an H-CDR3 as set forth in SEQ ID NO:17, and

(c) an antibody or antigen-binding portion thereof, comprising an H-CDR1as set forth in SEQ ID NO:29, an H-CDR2 as set forth in SEQ ID NO:30,and an H-CDR3 as set forth in SEQ ID NO:31.

In some further aspects, the disclosure provides an isolated antibody,or antigen-binding portion thereof, that specifically binds human 4-1BB,wherein said antibody or antigen-binding portion is selected from thegroup consisting of:

(a) an antibody or antigen-binding portion thereof, comprising an L-CDR1as set forth in SEQ ID NO:6, an L-CDR2 as set forth in SEQ ID NO:7, andan L-CDR3 as set forth in SEQ ID NO:8.

(b) an antibody or antigen-binding portion thereof, comprising an L-CDR1as set forth in SEQ ID NO:20, an L-CDR2 as set forth in SEQ ID NO:21,and an L-CDR3 as set forth in SEQ ID NO:22.

(c) an antibody or antigen-binding portion thereof, comprising an L-CDR1as set forth in SEQ ID NO:34, an L-CDR2 as set forth in SEQ ID NO:35,and an L-CDR3 as set forth in SEQ ID NO:36; and

(d) an antibody or antigen-binding portion thereof, comprising an L-CDR1as set forth in SEQ ID NO:34, an L-CDR2 as set forth in SEQ ID NO:35,and an L-CDR3 as set forth in SEQ ID NO:55.

In some further particular aspects, the isolated antibody is selectedfrom the group consisting of:

(a) an antibody or antigen-binding portion thereof, comprising: anH-CDR1 as set forth in SEQ ID NO:1, an H-CDR2 as set forth in SEQ IDNO:2, an H-CDR3 as set forth in SEQ ID NO:3; an L-CDR1 as set forth inSEQ ID NO:6, an L-CDR2 as set forth in SEQ ID NO:7, and an L-CDR3 as setforth in SEQ ID NO:8;

(b) an antibody or antigen-binding portion thereof, comprising an H-CDR1as set forth in SEQ ID NO:15, an H-CDR2 as set forth in SEQ ID NO:16, anH-CDR3 as set forth in SEQ ID NO:17; an L-CDR1 as set forth in SEQ IDNO:20, an L-CDR2 as set forth in SEQ ID NO:21, and an L-CDR3 as setforth in SEQ ID NO:22.

(c) an antibody or antigen-binding portion thereof, comprising an H-CDR1as set forth in SEQ ID NO:29, an H-CDR2 as set forth in SEQ ID NO:30, anH-CDR3 as set forth in SEQ ID NO:31; an L-CDR1 as set forth in SEQ IDNO:34, an L-CDR2 as set forth in SEQ ID NO:35, and an L-CDR3 as setforth in SEQ ID NO:36; and

(d) an antibody or antigen-binding portion thereof, comprising an H-CDR1as set forth in SEQ ID NO:29, an H-CDR2 as set forth in SEQ ID NO:30, anH-CDR3 as set forth in SEQ ID NO:31; an L-CDR1 as set forth in SEQ IDNO:34, an L-CDR2 as set forth in SEQ ID NO:35, and an L-CDR3 as setforth in SEQ ID NO:55.

In a further particular aspect, the isolated antibody comprises a V_(H)chain amino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:18, SEQID NO:32, and SEQ ID NO:43.

In a further particular aspect, the isolated antibody comprises a V_(L)chain amino acid sequence as set forth in SEQ ID NO:9, SEQ ID NO:23, SEQID NO:37, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:60, or SEQID NO:64.

In a further particular aspect, the isolated antibody comprises a V_(H)domain amino acid sequence as set forth in any one of SEQ ID NOs:4, 18,32, and :43, and further comprises a V_(L) domain amino acid sequence asset forth in any one of SEQ ID NOs:9, SEQ ID NO: 23, SEQ ID NO: 37, SEQID NO: 45, SEQ ID NO: 51, SEQ ID NO: 56, SEQ ID NO: 60, and SEQ ID NO:64.

In a further particular aspect, the isolated antibody is selected fromthe group consisting of:

(a) an antibody comprising a V_(H) chain amino acid sequence as setforth in SEQ ID NO:4 and a V_(L) chain amino acid sequence as set forthin SEQ ID NO:9;

(b) an antibody comprising a V_(H) chain amino acid sequence as setforth in SEQ ID NO:18 and a V_(L) chain amino acid sequence as set forthin SEQ ID NO:23;

(c) an antibody comprising a V_(H) chain amino acid sequence as setforth in SEQ ID NO:32 and a V_(L) chain amino acid sequence as set forthin SEQ ID NO:37 or SEQ ID NO 56; and

(d) an antibody comprising a V_(H) chain amino acid sequence as setforth in SEQ ID NO:43 and a V_(L) chain amino acid sequence as set forthin SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO: 60, or SEQ ID NO: 64.

In a still further particular aspect, the isolated antibody provided bythe present disclosure comprises a V_(H) chain that is encoded by (i) anucleic acid sequence comprising SEQ ID NO:11, SEQ ID NO:25, SEQ IDNO:39, SEQ ID NO:47, or (ii) a nucleic acid sequences that hybridizesunder high stringency conditions to the complementary strand of SEQ IDNO:11, SEQ ID NO:25, SEQ ID NO:39, or SEQ ID NO:47.

In a still further particular aspect, the isolated antibody comprises aV_(L) chain that is encoded by (i) a nucleic acid sequence comprisingSEQ ID NO:12, SEQ ID NO:26, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:53,SEQ ID NO:58, SEQ ID NO:62, or SEQ ID NO:66, or (ii) a nucleic acidsequences that hybridizes under high stringency conditions to thecomplementary strand of SEQ ID NO:12, SEQ ID NO:26, SEQ ID NO:40, SEQ IDNO:48, SEQ ID NO:53, SEQ ID NO:58, SEQ ID NO:62, or SEQ ID NO:66.

In a further particular aspect, there is provided an isolated antibodythat competes, and/or cross-competes for binding to human 4-1BB with anillustrative antibody selected from MOR-6032, MOR-7361, MOR-7480,MOR-7480.1, MOR-7480.2, MOR 7483, MOR-7483.1, or MOR-7483.2.

In a further particular aspect, there is provided an isolated antibodythat binds to the same epitope on human 4-1BB as any of the antibodiesdisclosed herein. In some embodiments, the disclosure provides anisolated antibody that binds to the same epitope on human 4-1BB as anillustrative antibody selected from MOR-6032, MOR-7361, MOR-7480,MOR-7480.1, MOR-7480.2, MOR 7483, MOR-7483.1, or MOR-7483.2.

In a further particular aspect, the present disclosure provides anisolated antibody that binds human 4-1BB, comprising a heavy chainvariable region that is the product of, or derived from, a human V_(H)3-23 gene, V_(H) 1-69 gene, or V_(H) 5. In another particular aspect thepresent disclosure provides an isolated antibody that binds human 4-1BB,comprising a light chain variable region that is the product of, orderived from, a human V_(L) λ3 or λ1-13 gene.

In some embodiments, the isolated antibodies described herein have oneor more of the following properties or characteristics:

a) specifically bind to human 4-1BB;

b) bind to human and cynomolgus 4-1BB;

c) bind to human 4-1BB or cynomolgus 4-1BB but not rat, or mouse 4-1BB;

d) are an IgG, such as IgG1, IgG2, IgG3, or IgG4; and

e) are human antibodies, or humanized antibodies.

In some other aspects, the present disclosure provides anantigen-binding portion of any of the antibody provided by the presentdisclosure. In some embodiments, the antigen-binding portion is Fab orscFv fragment.

In some further aspects, the present disclosure provides a derivative ofany of the antibodies provided by the present disclosure.

In some other aspects, the disclosure provides an isolated nucleic acidthat encodes a V_(H) chain of an antibody or antigen-binding portionthereof that bind human 4-1BB, which is selected from the groupconsisting of:

(i) a nucleic acid sequence that encodes a V_(H) chain amino acidsequence as set forth in SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:32, or SEQID NO:43;

(ii) a nucleic acid sequence as set forth in SEQ ID NO:11, SEQ ID NO:25,SEQ ID NO:39, or SEQ ID NO:47; or

(iii) a nucleic acid sequence that hybridizes under high stringencyconditions to the complementary strand of a nucleic acid sequence as setforth in SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:39, or SEQ ID NO:47.

In some other aspects, the disclosure provides an isolated nucleic acidthat encodes a V_(L) chain of an antibody or antigen-binding portionthereof that binds human 4-1BB, which is selected from the groupconsisting of:

(i) a nucleic acid sequence that encodes a V_(L) chain amino acidsequence as set forth in SEQ ID NO:9, SEQ ID NO:23, SEQ ID NO:37, SEQ IDNO:45, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:60, or SEQ ID NO:64;

(ii) a nucleic acid sequence as set forth in SEQ ID NO:12, SEQ ID NO:26,SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:58, SEQ ID NO: 62,or SEQ ID NO:66; or

(ii) a nucleic acid sequences that hybridizes under high stringencyconditions to the complementary strand of a nucleic acid sequence as setforth in SEQ ID NO:12, SEQ ID NO:26, SEQ ID NO:40, SEQ ID NO:48, SEQ IDNO:53, SEQ ID NO:58, SEQ ID NO: 62, or SEQ ID NO:66.

In some further aspects, the disclosure provides a vector comprising anyof the nucleic acids described herein. In a still further aspect, thedisclosure provides a host cell comprising any of the vectors describedherein. Such host cells can be bacterial or mammalian.

In some further aspects, the disclosure provides a pharmaceuticalcomposition comprising any of the antibodies, an antigen-bindingportions thereof, or a derivative thereof, and a pharmaceuticallyacceptable carrier.

The disclosure further provides methods for treating abnormal cellgrowth in a subject in need thereof, comprising administering to thesubject an effective amount of a binding molecule of the disclosure orpharmaceutical composition described herein. The disclosure furtherprovides methods of reducing tumor cell metastasis in a subject,comprising administering to said subject an effective amount of abinding molecule, or pharmaceutical compositions described herein.

In a further aspect, the disclosure provides a use of any of the bindingmolecules, or a pharmaceutical composition described herein, for themanufacture of a medicament for the treatment of abnormal cell growth ina subject in need thereof. In a further aspect, the disclosure providesa binding molecule, or a pharmaceutical composition, as describedherein, for use in the treatment of abnormal cell growth in a subject inneed thereof. In a yet further aspect, the disclosure provides a bindingmolecule, or a pharmaceutical composition, as described herein, for usein the treatment of tumor cell metastasis in a subject in need thereof.In a still further aspect, the disclosure provides a use of any of thebinding molecules, or a pharmaceutical composition described herein, forthe manufacture of a medicament for the treatment of tumor cellmetastasis in a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is four column graphs showing the mean fluorescence intensity ofunstimulated (black) and PHA stimulated (light grey) primary PBMC fromhuman (upper left), cynomolgus (upper right), dog (lower left), and rat(lower right) incubated with the indicated 4-1BB antibody or controlantibody conjugated to Alexafluor 647. The panel demonstrates binding tohuman and cynomolgus PBMC stimulated with PHA.

FIG. 2 is two line graphs showing luciferase reporter activity in 4-1BBexpressing 293T cells that have been stimulated with severalconcentrations of 4-1BB specific mAb or isotype control mAb. The leftpanel demonstrates reporter activity in cells that express cynomolgus4-1BB. The right panel demonstrates activity in cells that express human4-1BB. The data is expressed as fold stimulation above isotype control.

FIG. 3 (3A and 3B) are line graphs showing the concentration of humanIL-2 present in cell culture media following 72 hours of stimulation ofhuman T cells with anti-CD3 and several concentrations of 4-1BBantibodies. Each panel (A and B) represents an individual donor.

FIG. 4 is a scattergram showing the expansion of human peripheral bloodmononuclear cells in mice that have been treated with 4-1BB mAb orisotype control mAb. Data is expressed as the percentage of cellsexpressing human CD45 in the peripheral blood of individual NSG mice onstudy days 24-28 that had been injected with six million humanperipheral blood mononuclear cells on day 0 and injected with 1 mg/kg4-1BB mAb or isotype control mAb on day 9. Statistical significance wasdetermined using a two tailed Mann-Whitney test *p<0.05, **p<0.005. NoHBPT refers to animals that were not injected with human cells.

FIG. 5 is two column graphs showing the change in proliferating CD8central memory T cells at several time points following administrationof 4-1BB mAb in cynomolgus monkeys. Data is shown as columnsrepresenting individual animals designated as (dose level-animal number)and is represented as intra-animal change in the number of Ki-67+ cellsrelative to pre study number {[(#Ki-67+ cells on indicated studyday−#Ki-67+ cells at pre dose)/#Ki-67+ cells at pre dose]*100}. CD8central memory cells were identified as CD3+, CD8+, CD28+ and CD95+.

FIG. 6 are line graphs showing the growth of tumors injectedsubcutaneously with tumor cells (PC3, left panel; LOVO, right panel) andhuman peripheral blood mononuclear cells on study day 0. Mice wereinjected with 10 mg/kg of the indicated 4-1BB mAbs on day 0.

FIG. 7 Left panel is a scattergram showing the percentage of PBMC thatare positive for both the T cell surface marker CD8+ and haveincorporated the BrdU nucleoside analog following treatment of 4-1BBknock in mice with 4-1BB mAb or vehicle control. The right panel is aline graph showing the growth of murine melanoma tumors injectedsubcutaneously into 4-1BB knock in mice and treated with the indicatedconcentration of 4-1BB mAb.

FIG. 8 shows alignments of Amino Acid Sequences of the heavy ChainVariable Regions and Light Chain Variable Regions (with CDRs underlined)with Relevant Germline Sequences.

DETAILED DESCRIPTION A. Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The term “4-1BB antibody” refers to an antibody, as defined herein,capable of binding to human 4-1BB receptor.

The terms “4-1BB” and “4-1BB receptor” are used interchangeably in thepresent application, and include the human 4-1BB receptor, as well asvariants, isoforms, and species homologs thereof. Accordingly, a bindingmolecule, as defined and disclosed herein, may also bind 4-1BB fromspecies other than human. In other cases, a binding molecule may becompletely specific for the human 4-1BB and may not exhibit species orother types of cross-reactivity.

The articles “a” and “an” refer to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term “agonist” refers to a binding molecule, as defined herein,which upon binding to 4-1BB, (1) stimulates or activates 4-1BB, (2)enhances, increases, promotes, induces, or prolongs an activity,function, or presence of 4-1BB, or (3) enhances, increases, promotes, orinduces the expression of 4-1BB.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. The term “amino acidanalogs” refers to compounds that have the same basic chemical structureas a naturally occurring amino acid but the C-terminal carboxy group,the N-terminal amino group, or side chain functional group has beenchemically modified to another functional group. The term “amino acidmimetics” refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions similarly to a naturally occurring amino acid.

The term “antibody” is an art-recognized term and refers to anantigen-binding protein (i.e, immunoglobulin) having a basicfour-polypeptide chain structure consisting of two identical heavy (H)chains and two identical light (L) chains. Each L chain is linked to anH chain by one covalent disulfide bond, while the two H chains arelinked to each other by one or more disulfide bonds depending on the Hchain isotype. Each heavy chain has, at the N-terminus, a variableregion (abbreviated herein as V_(H)) followed by a constant region. Theheavy chain constant region is comprised of three domains, C_(H1),C_(H2) and C_(H3). Each light chain has, at the N-terminus, a variableregion (abbreviated herein as V_(I)) followed by a constant region atits other end. The light chain constant region is comprised of onedomain, C_(L). The V_(L) is aligned with the V_(H) and the C_(L) isaligned with the first constant domain of the heavy chain (CH₁). Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. An IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called J chain, and thereforecontains 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain.

The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). The CDR regions can be determined using the Kabat orChothia numbering systems, both of which are well known to those ofskill in the art. See, e.g. Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Chothia andLesk, J. Mol. Biol. 196:901-917 (1987). Each V_(H) and V_(L) is composedof three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Throughout the present disclosure, the three CDRs of theheavy chain are referred to as H-CDR1, H-CDR2, and H-CDR3. Similarly,the three CDRs of the light chain are referred to as L-CDR1, L-CDR2, andL-CDR3. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 ormore amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W.,ed., 2^(nd) ed. Raven Press, N.Y. (1989)).

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (CH), antibodiescan be assigned to different classes or isotypes. There are five classesof antibodies: IgA, IgD, IgE, IgG, and IgM, having heavy chainsdesignated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu),respectively. The IgG class of antibody can be further classified intofour subclasses IgG1, IgG2, IgG3, and IgG4 by the gamma heavy chains,Y1-Y4, respectively.

The term “antibody derivative” or “derivative” of an antibody refers toa molecule that is capable of binding to the same antigen (e.g., 4-1BB)that the antibody binds to and comprises an amino acid sequence of theantibody linked to an additional molecular entity. The amino acidsequence of the antibody that is contained in the antibody derivativemay be a full-length heavy chain, a full-length light chain, any portionor portions of a full-length heavy chain, any portion or portions of thefull-length light chain of the antibody, any other fragment(s) of anantibody, or the complete antibody. The additional molecular entity maybe a chemical or biological molecule. Examples of additional molecularentities include chemical groups, amino acids, peptides, proteins (suchas enzymes, antibodies), and chemical compounds. The additionalmolecular entity may have any utility, such as for use as a detectionagent, label, marker, pharmaceutical or therapeutic agent. The aminoacid sequence of an antibody may be attached or linked to the additionalmolecular entity by chemical coupling, genetic fusion, noncovalentassociation, or otherwise. The term “antibody derivative” alsoencompasses chimeric antibodies, humanized antibodies, and moleculesthat are derived from modifications of the amino acid sequences of a4-1BB antibody, such as conservation amino acid substitutions,additions, and insertions.

The term “antigen-binding fragment” or “antigen binding portion” of anantibody refers to one or more portions of an antibody that retain theability to bind to the antigen that the antibody bonds to (e.g., 4-1BB).Examples of “antigen-binding fragment” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H1) domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and C_(H1) domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb fragment (Ward et al., Nature 341:544-546(1989)), which consists of a V_(H) domain; and (vi) an isolatedcomplementarity determining region (CDR).

The term “binding molecule” encompasses (1) antibody, (2)antigen-binding fragment of an antibody, and (3) derivative of anantibody, each as defined herein.

The term “binding 4-1BB,” “binds 4-1BB,” “binding to 4-1BB,” or “bindsto 4-1BB” refers to the binding of a binding molecule, as definedherein, to the human 4-1BB in an in vitro assay, such as a BIAcore assayas described in Example 6, with an affinity (K_(D)) of 500 nM or less.

The term “chimeric antibody” refers to an antibody that comprises aminoacid sequences derived from different animal species, such as thosehaving a variable region derived from a human antibody and a murineimmunoglobulin constant region.

The term “compete for binding” refers to the interaction of twoantibodies in their binding to a binding target. A first antibodycompetes for binding with a second antibody if binding of the firstantibody with its cognate epitope is detectably decreased in thepresence of the second antibody compared to the binding of the firstantibody in the absence of the second antibody. The alternative, wherethe binding of the second antibody to its epitope is also detectablydecreased in the presence of the first antibody, can, but need not, bethe case. That is, a first antibody can inhibit the binding of a secondantibody to its epitope without that second antibody inhibiting thebinding of the first antibody to its respective epitope. However, whereeach antibody detectably inhibits the binding of the other antibody withits cognate epitope, whether to the same, greater, or lesser extent, theantibodies are said to “cross-compete” with each other for binding oftheir respective epitope(s).

The term “epitope” refers to a part of an antigen to which an antibody(or antigen-binding fragment thereof) binds. Epitopes can be formed bothfrom contiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope can include various numbers of aminoacids in a unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.(1996). Once a desired epitope on an antigen is determined, antibodiesto that epitope can be generated, e.g., using the techniques describedherein. The generation and characterization of antibodies may alsoelucidate information about desirable epitopes. From this information,it is then possible to competitively screen antibodies for binding tothe same epitope. An approach to achieve this is to conductcross-competition studies to find antibodies that competitively bindwith one another, i.e., the antibodies compete for binding to theantigen. A high throughput process for “binning” antibodies based upontheir cross-competition is described in PCT Publication No. WO 03/48731.

The term “germline” refers to the nucleotide sequences of the antibodygenes and gene segments as they are passed from parents to offspring viathe germ cells. The germline sequence is distinguished from thenucleotide sequences encoding antibodies in mature B cells which havebeen altered by recombination and hypermutation events during the courseof B cell maturation.

The term “glycosylation sites” refers to amino acid residues which arerecognized by a eukaryotic cell as locations for the attachment of sugarresidues. The amino acids where carbohydrate, such as oligosaccharide,is attached are typically asparagine (N-linkage), serine (O-linkage),and threonine (O-linkage) residues. The specific site of attachment istypically signaled by a sequence of amino acids, referred to herein as a“glycosylation site sequence”. The glycosylation site sequence forN-linked glycosylation is: -Asn-X-Ser- or -Asn-X-Thr-, where X may beany of the conventional amino acids, other than proline. The terms“N-linked” and “O-linked” refer to the chemical group that serves as theattachment site between the sugar molecule and the amino acid residue.N-linked sugars are attached through an amino group; O-linked sugars areattached through a hydroxyl group. The term “glycan occupancy” refers tothe existence of a carbohydrate moiety linked to a glycosylation site(i.e., the glycan site is occupied). Where there are at least twopotential glycosylation sites on a polypeptide, either none (0-glycansite occupancy), one (1-glycan site occupancy) or both (2-glycan siteoccupancy) sites can be occupied by a carbohydrate moiety.

The term “host cell” refers to a cellular system which can be engineeredto generate proteins, protein fragments, or peptides of interest. Hostcells include, without limitation, cultured cells, e.g., mammaliancultured cells derived from rodents (rats, mice, guinea pigs, orhamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissues orhybridoma cells, yeast cells, and insect cells, and cells comprisedwithin a transgenic animal or cultured tissue. The term encompasses notonly the particular subject cell but also the progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not beidentical to the parent cell, but are still included within the scope ofthe term “host cell.”

The term “human antibody” refers to an antibody in which the entireamino acid sequences of the light chains and heavy chains are from thehuman immunoglobulin genes. A human antibody may contain murinecarbohydrate chains if produced in a mouse, in a mouse cell or in ahybridoma derived from a mouse cell. Human antibodies may be prepared ina variety of ways known in the art.

The term “humanized antibody” refers to a chimeric antibody thatcontains amino acid residues derived from human antibody sequences. Ahumanized antibody may contain some or all of the CDRs from a non-humananimal antibody while the framework and constant regions of the antibodycontain amino acid residues derived from human antibody sequences.

The term “illustrative antibody” refers to any one of the antibodiesdescribed in the disclosure and designated as MOR-6032, MOR-7361,MOR-7480, MOR-7480.1, MOR-7480.2, MOR-7483, MOR-7483.1, and MOR-7483.2.These antibodies may be in any class (e.g., IgA, IgD, IgE, IgG, andIgM). Thus, each antibody identified above encompasses antibodies in allfive classes that have the same amino acid sequences for the V_(L) andV_(H) regions. Further, the antibodies in the IgG class may be in anysubclass (e.g., IgG1 IgG2, IgG3, and IgG4). Thus, each antibodyidentified above in the IgG subclass encompasses antibodies in all foursubclasses that have the same amino acid sequences for the V_(L) andV_(H) regions. The amino acid sequences of the heavy chain constantregions of human antibodies in the five classes, as well as in the fourIgG subclasses, are known in the art. As examples, the amino acidsequences of the human IgG1 and IgG2 constant regions are provided inSEQ ID NOs: 69 and 71, respectively. The amino acid sequence of the fulllength heavy chain for the IgG2 subclass of each of the illustrativeantibodies is provided in the disclosure.

The term “isolated antibody” or “isolated binding molecule” refers to anantibody or a binding molecule, as defined herein, that: (1) is notassociated with naturally associated components that accompany it in itsnative state; (2) is free of other proteins from the same species; (3)is expressed by a cell from a different species; or (4) does not occurin nature. Examples of isolated antibodies include a 4-1BB antibody thathas been affinity purified using 4-1BB, a 4-1BB antibody that has beengenerated by hybridomas or other cell line in vitro, and a 4-1BBantibody derived from a transgenic animal.

The term “isolated nucleic acid” refers to a nucleic acid molecule ofgenomic, cDNA, or synthetic origin, or a combination thereof, which isseparated from other nucleic acid molecules present in the naturalsource of the nucleic acid. For example, with regard to genomic DNA, theterm “isolated” includes nucleic acid molecules which are separated fromthe chromosome with which the genomic DNA is naturally associated.Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid of interest.

The term “k_(a)” refers to the association rate constant of a particularantibody-antigen interaction, whereas the term “k_(d)” refers to thedissociation rate constant of a particular antibody-antigen interaction.

The term “K_(D)” refers to the equilibrium dissociation constant of aparticular antibody-antigen interaction. It is obtained from the ratioof k_(d) to k_(a) (i.e., k_(d)/k_(a)) and is expressed as a molarconcentration (M). K_(D) is used as a measure for the affinity of anantibody's binding to its binding partner. The smaller the K_(D), themore tightly bound the antibody is, or the higher the affinity betweenantibody and the antigen. For example, an antibody with a nanomolar (nM)dissociation constant binds more tightly to a particular antigen than anantibody with a micromolar (μM) dissociation constant. K_(D) values forantibodies can be determined using methods well established in the art.One method for determining the K_(D) of an antibody is by using surfaceplasmon resonance, typically using a biosensor system such as a Biacore®system. An assay procedure using the BIACORE™ system (BIAcore assay) isdescribed in the Examples section of this disclosure.

The term “mammal” refers to any animal species of the Mammalia class.Examples of mammals include: humans; laboratory animals such as rats,mice, simians and guinea pigs; domestic animals such as cats, dogs,rabbits, cattle, sheep, goats, horses, and pigs; and captive wildanimals such as lions, tigers, elephants, and the like.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to polyclonal antibodypreparations which include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen. In addition to theirspecificity, the monoclonal antibodies are advantageous in that they maybe synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies may be prepared by the hybridoma methodology or may be madeusing recombinant DNA methods in bacterial, eukaryotic animal or plantcells (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies mayalso be isolated from phage antibody libraries using the techniquesdescribed in Clackson et al., Nature, 352:624-628 (1991) and Marks etal., J. Mol. Biol., 222:581-597 (1991), for example.

The term “prevent” or “preventing,” with reference to a certain diseasecondition in a mammal, refers to preventing or delaying the onset of thedisease, or preventing the manifestation of clinical or subclinicalsymptoms thereof.

The term “recombinant antibody” refers to an antibody that is prepared,expressed, created or isolated by recombinant means, such as antibodiesisolated from an animal that is transgenic for another species'immunoglobulin genes, antibodies expressed using a recombinantexpression vector transfected into a host cell, antibodies isolated froma recombinant, combinatorial antibody library, or antibodies prepared,expressed, created or isolated by any other means that involves splicingof immunoglobulin gene sequences to other DNA sequences.

As used herein, “sequence identity” between two polypeptide sequencesindicates the percentage of amino acids that are identical between thesequences. The amino acid sequence identity of polypeptides can bedetermined conventionally using known computer programs such as Bestfit,FASTA, or BLAST (see, e.g. Pearson, Methods Enzymol. 183:63-98 (1990);Pearson, Methods Mol. Biol. 132:185-219 (2000); Altschul et al., J. Mol.Biol. 215:403-410 (1990); Altschul et al., Nucelic Acids Res.25:3389-3402 (1997)). When using Bestfit or any other sequence alignmentprogram to determine whether a particular sequence is, for instance, 95%identical to a reference amino acid sequence, the parameters are setsuch that the percentage of identity is calculated over the full lengthof the reference amino acid sequence and that gaps in homology of up to5% of the total number of amino acid residues in the reference sequenceare allowed. This aforementioned method in determining the percentage ofidentity between polypeptides is applicable to all proteins, fragments,or variants thereof disclosed herein.

The term “specifically binds” or “specifically binds to,” in referenceto the interaction of a binding molecule, as defined herein, (e.g., anantibody) with its binding partner (e.g., an antigen), refers to theability of the binding molecule to discriminate between an antigen ofinterest from an animal species and the antigen orthologue from adifferent animal species under a given set of conditions. A 4-1BBbinding molecule is said to specifically bind to human 4-1BB if it bindsto human 4-1BB at an EC50 that is below 50 percent of the EC50 at whichit binds 4-1BB of rat or mouse as determined in an in vitro assay.Binding specificity of an antibody can be determined using methods knownin the art. Examples of such methods include FACS using PHA stimulatedprimary cells, Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptidescans.

The term “selectively binds” or “selectively binds to,” in reference tothe interaction of a binding molecule, as defined herein, (e.g., anantibody) with its binding partner (e.g., an antigen), refers to theability of the binding molecule to discriminate between an antigen ofinterest from an animal species (such as human 4-1BB) and a differentantigen from the same animal species (such as human CD40) under a givenset of conditions. A 4-1BB binding molecule is said to selectively bindto human 4-1BB if it binds to human 4-1BB at an EC50 that is below 10percent of the EC50 at which it binds to human CD40 or human CD134 asdetermined in an in vitro assay.

The term “treat”, “treating”, or “treatment”, with reference to acertain disease condition in a mammal, refers causing a desirable orbeneficial effect in the mammal having the disease condition. Thedesirable or beneficial effect may include reduced frequency or severityof one or more symptoms of the disease (i.e., tumor growth and/ormetastasis, or other effect mediated by the numbers and/or activity ofimmune cells, and the like), or arrest or inhibition of furtherdevelopment of the disease, condition, or disorder. In the context oftreating cancer in a mammal, the desirable or beneficial effect mayinclude inhibition of further growth or spread of cancer cells, death ofcancer cells, inhibition of reoccurrence of cancer, reduction of painassociated with the cancer, or improved survival of the mammal. Theeffect can be either subjective or objective. For example, if the mammalis human, the human may note improved vigor or vitality or decreasedpain as subjective symptoms of improvement or response to therapy.Alternatively, the clinician may notice a decrease in tumor size ortumor burden based on physical exam, laboratory parameters, tumormarkers or radiographic findings. Some laboratory signs that theclinician may observe for response to treatment include normalization oftests, such as white blood cell count, red blood cell count, plateletcount, erythrocyte sedimentation rate, and various enzyme levels.Additionally, the clinician may observe a decrease in a detectable tumormarker. Alternatively, other tests can be used to evaluate objectiveimprovement, such as sonograms, nuclear magnetic resonance testing andpositron emissions testing.

The term “vector” refers to a nucleic acid molecule capable oftransporting a foreign nucleic acid molecule. The foreign nucleic acidmolecule is linked to the vector nucleic acid molecule by a recombinanttechnique, such as ligation or recombination. This allows the foreignnucleic acid molecule to be multiplied, selected, further manipulated orexpressed in a host cell or organism. A vector can be a plasmid, phage,transposon, cosmid, chromosome, virus, or virion. One type of vectorscan be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome(e.g., non-episomal mammalian vectors). Another type of vectors arecapable of autonomous replication in a host cell into which it isintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Another specific type ofvectors capable of directing the expression of expressible foreignnucleic acids to which they are operatively linked are commonly referredto “expression vectors.” Expression vectors generally have controlsequences that drive expression of the expressible foreign nucleicacids. Simpler vectors, known as “transcription vectors,” are onlycapable of being transcribed but not translated: they can be replicatedin a target cell but not expressed. The term “vector” encompasses alltypes of vectors regardless of their function. Vectors capable ofdirecting the expression of expressible nucleic acids to which they areoperatively linked are commonly referred to “expression vectors.”

The methods and techniques of the present disclosure are generallyperformed according to methods well known in the art and as described invarious general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Such references include, e.g., Sambrook and Russell,Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (2001), Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, NY (2002), and Harlow and LaneAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990). Enzymatic reactions and purificationtechniques are performed according to manufacturer's specifications, ascommonly accomplished in the art or as described herein. Thenomenclatures used in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)).

B. Binding Molecules that Bind to Human 4-1BB

The present disclosure provides isolated binding molecules that bind tohuman 4-1BB, including 4-1BB antibodies, antigen-binding fragments ofthe 4-1BB antibodies, and derivatives of the 4-1BB antibodies.

B-1. 4-1BB Antibodies

In some aspects, the present disclosure provides an isolated antibodythat binds to human 4-1BB at an epitope within amino acid residues115-156 of SEQ ID No: 68. In some embodiments, the isolated antibodycomprises the H-CDR1 amino acid sequence of SEQ ID NO: 29, H-CDR2 aminoacid sequence of SEQ ID NO: 30, and H-CDR3 amino acid sequence of SEQ IDNO: 31. In some other embodiments, the isolated antibody comprises theL-CDR1 amino acid sequence of SEQ ID NO: 34, L-CDR2 amino acid sequenceof SEQ ID NO: 35, and L-CDR3 amino acid sequence of SEQ ID NO: 36. Insome other embodiments, the antibodies described herein above have oneor more biological properties described herein below.

In other aspects, the disclosure provides an isolated antibody thatbinds to human 4-1BB, wherein said antibody comprises: (a) an H-CDR1 asset forth in SEQ ID NO:1, SEQ ID NO:15, or SEQ ID NO:29; (b) an H-CDR2as set forth in SEQ ID NO:2, SEQ ID NO:16, or SEQ ID NO:30; and (c) anH-CDR3 as set forth in SEQ ID NO:3, SEQ ID NO: 17, or SEQ ID NO:31.

In another aspect, the disclosure provides an isolated antibody thatbinds to human 4-1BB, wherein said antibody comprises: (a) an L-CDR1 asset forth in SEQ ID NO:6, SEQ ID NO: 20, or SEQ ID NO:34; (b) an L-CDR2as set forth in SEQ ID NO:7, SEQ ID NO:21, or SEQ ID NO:35; and (c) anL-CDR3 as set forth in SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:36, or SEQID NO: 55.

In a further aspect, the disclosure provides an isolated antibody thatbinds to human 4-1BB, wherein said antibody comprises: (a) an H-CDR1 asset forth in SEQ ID NO:1, SEQ ID NO:15, or SEQ ID NO:29; (b) an H-CDR2as set forth in SEQ ID NO:2, SEQ ID NO:16, or SEQ ID NO:30; and (c) anH-CDR3 as set forth in SEQ ID NO:3, SEQ ID NO: 17, or SEQ ID NO:31; andfurther comprises: (d) an L-CDR1 as set forth in SEQ ID NO:6, SEQ ID NO:20, or SEQ ID NO:34; (e) an L-CDR2 as set forth in SEQ ID NO:7, SEQ IDNO:21, or SEQ ID NO:35; and (f) an L-CDR3 as set forth in SEQ ID NO:8,SEQ ID NO:22, SEQ ID NO:36, or SEQ ID NO: 55.

In some further aspects, the disclosure provides an isolated antibodythat binds to human 4-1BB, wherein said antibody is selected from thegroup consisting of:

(a) an antibody comprising an H-CDR1 as set forth in SEQ ID NO:1, anH-CDR2 as set forth in SEQ ID NO:2, and an H-CDR3 as set forth in SEQ IDNO:3;

(b) an antibody comprising an H-CDR1 as set forth in SEQ ID NO:15, anH-CDR2 as set forth in SEQ ID NO:16, and an H-CDR3 as set forth in SEQID NO:17; and

(c) an antibody comprising an H-CDR1 as set forth in SEQ ID NO:29, anH-CDR2 as set forth in SEQ ID NO:30, and an H-CDR3 as set forth in SEQID NO:31.

In some further aspects, the disclosure provides an isolated antibodythat binds to human 4-1BB, wherein said antibody is selected from thegroup consisting of:

(a) an antibody comprising an L-CDR1 as set forth in SEQ ID NO:6, anL-CDR2 as set forth in SEQ ID NO:7, and an L-CDR3 as set forth in SEQ IDNO:8;

(b) an antibody comprising an L-CDR1 as set forth in SEQ ID NO:20, anL-CDR2 as set forth in SEQ ID NO:21, and an L-CDR3 as set forth in SEQID NO:22;

(c) an antibody comprising an L-CDR1 as set forth in SEQ ID NO:34, anL-CDR2 as set forth in SEQ ID NO:35, and an L-CDR3 as set forth in SEQID NO:36; and

(d) an antibody comprising an L-CDR1 as set forth in SEQ ID NO:34, anL-CDR2 as set forth in SEQ ID NO:35, and an L-CDR3 as set forth in SEQID NO:55.

In some further aspects, the disclosure provides an isolated antibodythat binds to the human 4-1BB, wherein said antibody is selected fromthe group consisting of:

(a) an antibody comprising an H-CDR1 as set forth in SEQ ID NO:1, anH-CDR2 as set forth in SEQ ID NO:2, an H-CDR3 as set forth in SEQ IDNO:3; an L-CDR1 as set forth in SEQ ID NO:6, an L-CDR2 as set forth inSEQ ID NO:7, and an L-CDR3 as set forth in SEQ ID NO:8;

(b) an antibody comprising an H-CDR1 as set forth in SEQ ID NO:15, anH-CDR2 as set forth in SEQ ID NO:16, an H-CDR3 as set forth in SEQ IDNO:17; an L-CDR1 as set forth in SEQ ID NO:20, an L-CDR2 as set forth inSEQ ID NO:21, and an L-CDR3 as set forth in SEQ ID NO:22;

(c) an antibody comprising an H-CDR1 as set forth in SEQ ID NO:29, anH-CDR2 as set forth in SEQ ID NO:30, an H-CDR3 as set forth in SEQ IDNO:31; an L-CDR1 as set forth in SEQ ID NO:34, an L-CDR2 as set forth inSEQ ID NO:35, and an L-CDR3 as set forth in SEQ ID NO:36; and

(d) an antibody comprising an H-CDR1 as set forth in SEQ ID NO:29, anH-CDR2 as set forth in SEQ ID NO:30, an H-CDR3 as set forth in SEQ IDNO:31; an L-CDR1 as set forth in SEQ ID NO:34, an L-CDR2 as set forth inSEQ ID NO:35, and an L-CDR3 as set forth in SEQ ID NO:55.

In a further aspect, the disclosure provides an isolated antibody thatbinds to human 4-1BB, wherein said antibody comprises a V_(H) chainamino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:18, SEQ IDNO:32, or SEQ ID NO:43.

In a further aspect, the disclosure provides an isolated antibody thatbinds to human 4-1BB, wherein said antibody comprises a V_(L) chainamino acid sequence as set forth in SEQ ID NO:9, SEQ ID NO:23, SEQ IDNO:37, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:60, or SEQ IDNO:64.

In a further aspect, the disclosure provides an isolated antibody thatbinds to human 4-1BB, wherein said antibody comprises (1) a V_(H) chainamino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:18, SEQ IDNO:32, or SEQ ID NO:43, and (2) a V_(L) chain amino acid sequence as setforth in SEQ ID NO:9, SEQ ID NO:23, SEQ ID NO:37, SEQ ID NO:45, SEQ IDNO:51, SEQ ID NO:56, SEQ ID NO:60, or SEQ ID NO:64.

In a further aspect, the disclosure provides an isolated antibody thatbinds to human 4-1BB, wherein said antibody is selected from the groupconsisting of:

(a) an antibody comprising a V_(H) chain amino acid sequence as setforth in SEQ ID NO:4 and a V_(L) chain amino acid sequence as set forthin SEQ ID NO:9;

(b) an antibody comprising a V_(H) chain amino acid sequence as setforth in SEQ ID NO:18 and a V_(L) chain amino acid sequence as set forthin SEQ ID NO:23;

(c) an antibody comprising a V_(H) chain amino acid sequence as setforth in SEQ ID NO:32 and a V_(L) chain amino acid sequence as set forthin SEQ ID NO:37 or SEQ ID NO 56; and

(d) an antibody comprising a V_(H) chain amino acid sequence as setforth in SEQ ID NO:43 and a V_(L) chain amino acid sequence as set forthin SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO: 60, or SEQ ID NO: 64.

In some embodiments, the antibodies described herein above, includingantibodies described with reference to epitope binding and antibodiesdescribed with reference to specific amino acid sequences of CDRs orvariable regions, have at least one of the following functionalproperties: (a) bind to human 4-1BB with a K_(D) of 500 nM or less; (b)have agonist activity on human 4-1BB; (c) do not bind to human CD40receptor at concentration up to 1000 nM; (d) do not bind to human CD134receptor at concentrations up to 1000 nM; (e) do not bind to rat, ormouse 4-1BB at concentrations up to 100 nM; (h) are capable ofinhibiting tumor cell growth; and (i) have therapeutic effect on acancer. In some further embodiments, the antibodies specifically bind tohuman 4-1BB with a K_(D) of 500 nM or less, 100 nM or less, 50 nM orless, 10 nM or less, 5 nM or less, or 1 nM or less, for the human 4-1BBextracellular domain as measured with the BIACore assay described inthis disclosure. In still further embodiments, the antibody is a humanantibody or humanized antibody that specifically binds to human 4-1BBwith a K_(D) of 500 nM or less, 100 nM or less, 50 nM or less, 10 nM orless, 5 nM or less, or 1 nM or less, for the human 4-1BB extracellulardomain as measured with the BIACore assay described in this disclosure.In some further embodiments, the antibody is a human antibody thatspecifically and selectively binds to human 4-1BB.

In other embodiments, the antibodies described herein above comprise aheavy chain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene, such as an antibodycomprising a heavy chain variable region that is the product of, orderived from, a human V_(H) 1-69 gene, V_(H) 3-23 gene, or V_(H) 5 gene.Exemplary antibodies include MOR-7480.1, MOR-7480.2, MOR-7483.1, andMOR-7483.2, each of which contains amino acids that derived from humangermline V_(H)5 gene.

In still other embodiments, the antibodies described herein abovecomprise a light chain variable region that is derived from a humanV_(L) λ3 gene. In yet other embodiment the antibodies described hereinabove comprise a heavy chain variable region that is the product of, orderived from, a human V_(H) 1-69 gene, V_(H) 3-23 gene, or V_(H) 5 gene,and further comprise a light chain variable region that is the productof, or derived from, a human V_(L) λ3 gene, wherein the antibody orportion thereof specifically binds to human 4-1BB. Exemplary antibodiesinclude MOR-7480.1, MOR-7480.2, MOR-7483.1, and MOR-7483.2, each ofwhich contains amino acids that derived from human germline V_(H)5 geneand V_(L) λ3 gene, respectively.

As used herein, a human antibody comprises heavy or light chain variableregions that is “derived from” a particular germline sequence if thevariable regions of the antibody are obtained from a system that useshuman germline immunoglobulin genes. Such systems include immunizing atransgenic mouse carrying human immunoglobulin genes with the antigen ofinterest or screening a human immunoglobulin gene library displayed onphage with the antigen of interest. A human antibody that is “derivedfrom” a human germline immunoglobulin sequence can be identified as suchby comparing the amino acid sequence of the human antibody to the aminoacid sequences of human germline immunoglobulins and selecting the humangermline immunoglobulin sequence that is closest in sequence (i.e.,greatest % identity) to the sequence of the human antibody. A humanantibody that is “derived from” a particular human germlineimmunoglobulin sequence may contain amino acid differences as comparedto the germline sequence, due to, for example, naturally-occurringsomatic mutations or intentional introduction of site-directed mutation.However, a selected human antibody typically is at least 90% identicalin amino acid sequence to an amino acid sequence encoded by a humangermline immunoglobulin gene and contains amino acid residues thatidentify the human antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. In certain cases, the human antibody is identicalin amino acid sequence to the amino acid sequence encoded by thegermline Ig gene. Typically, a human antibody derived from a particularhuman germline sequence will display no more than 10 amino aciddifferences from the amino acid sequence encoded by the human germlineimmunoglobulin gene. In certain cases, the human antibody may display nomore than 5, or even no more than 4, 3, 2, or 1 amino acid differencesfrom the amino acid sequence encoded by the germline immunoglobulingene. Alignments of the amino acid sequences of variable regions of theillustrative antibodies and the relevant germlines are provided in FIG.6.

In another aspect, the disclosure provides isolated antibodies thatcompete or cross-compete for binding to human 4-1BB with any of theillustrative antibodies of the disclosure, such as MOR-6032, MOR-7361,MOR-7480, MOR-7480.1, MOR-7480.2, MOR-7483, MOR-7483.1, or MOR-7483.2.In a particular embodiment, the disclosure provides isolated antibodiesthat compete or cross-compete for binding to the same epitope on thehuman 4-1BB with any of the illustrative antibodies of the disclosure.The ability of an antibody to compete cross-compete for binding withanother antibody can be determined using standard binding assays knownin the art, such as BIAcore analysis, ELISA assays, or flow cytometry.For example, one can allow an illustrative antibody of the disclosure tobind to human 4-1BB under saturating conditions and then measure theability of the test antibody to bind to the 4-1BB. If the test antibodyis able to bind to the 4-1BB at the same time as the illustrativeantibody, then the test antibody binds to a different epitope as theillustrative antibody. However, if the test antibody is not able to bindto the 4-1BB at the same time, then the test antibody binds to the sameepitope, an overlapping epitope, or an epitope that is in closeproximity to the epitope bound by the illustrative antibody. Thisexperiment can be performed using various methods, such as ELISA, RIA,FACS or surface plasmon resonance.

The 4-1BB antibodies described herein can be in any class, such as IgG,IgM, IgE, IgA, or IgD. It is preferred that the 4-1BB antibodies are inthe IgG class, such as IgG1, IgG2, IgG3, or IgG4 subclass, morepreferably IgG2 subclass. A 4-1BB antibody can be converted from oneclass or subclass to another class or subclass using methods known inthe art. An exemplary method for producing an antibody in a desiredclass or subclass comprises the steps of isolating a nucleic acidencoding a heavy chain of an 4-1BB antibody and a nucleic acid encodinga light chain of a 4-1BB antibody, isolating the sequence encoding theV_(H) region, ligating the V_(H) sequence to a sequence encoding a heavychain constant region of the desired class or subclass, expressing thelight chain gene and the heavy chain construct in a cell, and collectingthe 4-1BB antibody.

Further, the antibodies provided by the present disclosure can bemonoclonal or polyclonal, but preferably monoclonal.

Examples of specific isolated antibodies provided by the presentdisclosure include the following illustrative antibodies: MOR-6032,MOR-7361, MOR-7480, MOR-7480.1, MOR-7480.1, MOR-7480.2, MOR-7483,MOR-7483, MOR-7483.1, and MOR-7483.2. The nucleotide and amino acidsequences of the heavy chain variable region, full length heavy chainfor the IgG2 subclass, light chain variable region, and full lengthlight chain of these antibodies are provided in this disclosure; anindex of the SEQ ID NOs for these sequences is provided in Table 1. Theamino acid sequences of the CDRs of these illustrative antibodies areshown in Table 2.

TABLE 1 Index of SEQ ID NOs Full length Amino Variable Region AcidNucleotide Amino Acid Nucleotide SEQ ID SEQ ID SEQ ID SEQ ID AntibodyChain NO NO NO NO MOR-6032 Heavy 5 13 4 11 Light 10 14 9 12 MOR-7361Heavy 19 27 18 25 Light 24 28 23 26 MOR-7480 Heavy 33 41 32 39 Light 3842 37 40 MOR-7480.1 Heavy 44 49 43 47 Light 46 50 45 48 MOR-7480.2 Heavy44 49 43 47 Light 52 54 51 53 MOR-7483 Heavy 33 41 32 39 Light 57 59 5658 MOR-7483.1 Heavy 44 49 43 47 Light 61 63 60 62 MOR-7483.2 Heavy 44 4943 47 Light 65 67 64 66

TABLE 2 Amino Acid Sequence of CDRs Antibody CDR Sequence SEQ ID NOMOR-6032 H-CDR1 NSYAIS 1 H-CDR2 GIIPGFGTANYAQKFQG 2 H-CDR3 RKNEEDGGFDH 3L-CDR1 SGDNLGDYYAS 6 L-CDR2 DDSNRPS 7 L-CDR3 QTWDGTLHFV 8 MOR-7361H-CDR1 SDYYMH 15 H-CDR2 VISGSGSNTYYADSVKG 16 H-CDR3 RLYAQFEGDF 17 L-CDR1SGDNIGSKYVS 20 L-CDR2 SDSERPS 21 L-CDR3 QSWDGSISRV 22 MOR-7480; H-CDR1STYWIS 29 MOR-7480.1; H-CDR2 KIYPGDSYTNYSPSFQG 30 MOR-7480.2 H-CDR3RGYGIFDY 31 L-CDR1 SGDNIGDQYAH 34 L-CDR2 QDKNRPS 35 L-CDR3 ATYTGFGSLAV36 MOR-7483; H-CDR1 STYWIS 29 MOR-7483.1; H-CDR2 KIYPGDSYTNYSPSFQG 30MOR-7483.2 H-CDR3 RGYGIFDY 31 L-CDR1 SGDNIGDQYAH 34 L-CDR2 QDKNRPS 35L-CDR3 STYTFVGFTTV 55

Antibodies of the present disclosure can be produced by techniques knownin the art, including conventional monoclonal antibody methodology e.g.,the standard somatic cell hybridization technique (See e.g., Kohler andMilstein, Nature 256:495 (1975), viral or oncogenic transformation of Blymphocytes, or recombinant antibody technologies as described in detailherein below.

Hybridoma production is a very well-established procedure. The commonanimal system for preparing hybridomas is the murine system.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known. Onewell-known method that may be used for making human 4-1BB antibodiesprovided by the present disclosure involves the use of a XenoMouse™animal system. XenoMouse™ mice are engineered mouse strains thatcomprise large fragments of human immunoglobulin heavy chain and lightchain loci and are deficient in mouse antibody production. See, e.g.,Green et al., Nature Genetics 7:13-21 (1994) and WO2003/040170. Theanimal is immunized with a 4-1BB antigen. The 4-1BB antigen is isolatedand/or purified 4-1BB, preferably 4-1BB. It may be a fragment of 4-1BB,such as the extracellular domain of 4-1BB, particularly a 4-1BBextracellular domain fragment comprising amino acid resides 115-156 ofSEQ ID NO: 68. Immunization of animals can be carried out by any methodknown in the art. See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, New York: Cold Spring Harbor Press, 1990. Methods for immunizingnon-human animals such as mice, rats, sheep, goats, pigs, cattle andhorses are well known in the art. See, e.g., Harlow and Lane, supra, andU.S. Pat. No. 5,994,619. The 4-1BB antigen may be administered with anadjuvant to stimulate the immune response. Exemplary adjuvants includecomplete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) orISCOM (immunostimulating complexes). After immunization of an animalwith a 4-1BB antigen, antibody-producing immortalized cell lines areprepared from cells isolated from the immunized animal. Afterimmunization, the animal is sacrificed and lymph node and/or splenic Bcells are immortalized. Methods of immortalizing cells include, but arenot limited to, transferring them with oncogenes, inflecting them withthe oncogenic virus cultivating them under conditions that select forimmortalized cells, subjecting them to carcinogenic or mutatingcompounds, fusing them with an immortalized cell, e.g., a myeloma cell,and inactivating a tumor suppressor gene. See, e.g., Harlow and Lane,supra. If fusion with myeloma cells is used, the myeloma cellspreferably do not secrete immunoglobulin polypeptides (a non-secretorycell line). Immortalized cells are screened using 4-1BB, a portionthereof, or a cell expressing 4-1BB. 4-1BB antibody-producing cells,e.g., hybridomas, are selected, cloned and further screened fordesirable characteristics, including robust growth, high antibodyproduction and desirable antibody characteristics, as discussed furtherbelow. Hybridomas can be expanded in vivo in syngeneic animals, inanimals that lack an immune system, e.g., nude mice, or in cell culturein vitro. Methods of selecting, cloning and expanding hybridomas arewell known to those of ordinary skill in the art.

Antibodies of the disclosure can also be prepared using phage displaymethods.

Such phage display methods for isolating human antibodies areestablished in the art, such as the HuCAL® Libraries as describedfurther in Example 1. See also, for example: Achim Knappik, et al: FullySynthetic Human Combinatorial Antibody Libraries (HuCAL) Based onModular Consensus Frameworks and CDRs Randomized with Trinucleotides. J.Mol. Biol. (2000) 296, 57-86.

B-2. Antigen-Binding Fragments

In some other aspects, the present disclosure provides antigen-bindingfragments of any of the 4-1BB antibodies provided by the presentdisclosure.

The antigen-binding fragment may comprise any sequences of the antibody.In some embodiments, the antigen-binding fragment comprises the aminoacid sequence of: (1) a light chain of a 4-1BB antibody; (2) a heavychain of a 4-1BB antibody; (3) a variable region from the light chain ofa 4-1BB antibody; (4) a variable region from the heavy chain of a 4-1BBantibody; (5) one or more CDRs (two, three, four, five, or six CDRs) ofa 4-1BB antibody; or (6) three CDRs from the light chain and three CDRsfrom the heavy chain of a 4-1BB antibody.

In some particular embodiments, the disclosure provides anantigen-binding fragment of an antibody selected from: MOR-6032,MOR-7361, MOR-7480, MOR-7480.1, MOR-7480.2, MOR-7483, MOR-7483.1, orMOR-7483.2.

In some other particular embodiments, the antigen-binding fragments ofan 4-1BB antibody include: (i) a Fab fragment, which is a monovalentfragment consisting of the V_(L), V_(H), C_(L) and C_(H)1 domains; (ii)a F(ab′)₂ fragment, which is a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H)1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; (vi) an isolated CDR, and (vii) singlechain antibody (scFv), which is a polypeptide comprising a V_(L) regionof an antibody linked to a V_(H) region of an antibody. Bird et al.,(1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883.

In some particular embodiments, the antigen-binding fragment is a Fabfragment selected from the group consisting of Fab-6032, Fab-7361,Fab-7480, and Fab-7483.

B-3. Antibody Derivatives

In some further aspects, the present disclosure provides derivatives ofany of the 4-1BB antibodies provided by the present disclosure.

In one aspect, the antibody derivative is derived from modifications ofthe amino acid sequences of an illustrative antibody (“parent antibody”)of the disclosure while conserving the overall molecular structure ofthe parent antibody amino acid sequence. Amino acid sequences of anyregions of the parent antibody chains may be modified, such as frameworkregions, CDR regions, or constant regions. Types of modificationsinclude substitutions, insertions, deletions, or combinations thereof,of one or more amino acids of the parent antibody. In some embodiments,the antibody derivative comprises an V_(H) region that is at least 65%,at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to an amino acidsequence as set forth in any of SEQ ID NOs: 4, 18, 32, or 43. In someother embodiments, the antibody derivative comprises an V_(L) regionthat is at least 65%, at least 75%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto an amino acid sequence as set forth in any of SEQ ID NOs: 9, 23, 37,45, 51, 56, 60, or 64. In some particular embodiments, the derivativecomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15conservative or non-conservative substitutions, and/or 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and/or deletions to anamino acid sequence as set forth in any of SEQ ID NOs: 4, 18, 32, 43, 9,23, 37, 45, 51, 56, 60, or 64.

Amino acid substitutions encompass both conservative substitutions andnon-conservative substitutions. The term “conservative amino acidsubstitution” means a replacement of one amino acid with another aminoacid where the two amino acids have similarity in certainphysico-chemical properties such as polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, substitutions typically may be madewithin each of the following groups: (a) nonpolar (hydrophobic) aminoacids, such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine; (b) polar neutral aminoacids, such as glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; (c) positively charged (basic) amino acids,such as arginine, lysine, and histidine; and (d) negatively charged(acidic) amino acids, such as aspartic acid and glutamic acid.

The modifications may be made in any positions of the amino acidsequences of the antibody, including the CDRs, framework regions, orconstant regions. In one embodiment, the present disclosure provides anantibody derivative that contains the V_(H) and V_(L) CDR sequences ofan illustrative antibody of this disclosure, yet contains frameworksequences different from those of the illustrative antibody. Suchframework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the Genbank database or in the “VBase”human germline sequence database (Kabat, E. A., et al., Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242 (1991);Tomlinson, I. M., et al., J. Mol. Biol. 227:776-798 (1992); and Cox, J.P. L. et al., Eur. J. Immunol. 24:827-836 (1994)). Framework sequencesthat may be used in constructing an antibody derivative include thosethat are structurally similar to the framework sequences used byillustrative antibodies of the disclosure, e.g., similar to the V_(H)3-23 framework sequences and/or the V_(L) λ3 or λ1-13 frameworksequences used by illustrative antibodies of the disclosure. Forexample, the H-CDR1, H-CDR2, and H-CDR3 sequences, and the L-CDR1,L-CDR2, and L-CDR3 sequences of an illustrative antibody can be graftedonto framework regions that have the identical sequence as that found inthe germline immunoglobulin gene from which the framework sequencederive, or the CDR sequences can be grafted onto framework regions thatcontain one or more mutations as compared to the germline sequences.

In a particular embodiment, the antibody derivative is a chimericantibody which comprises an amino acid sequence of an illustrativeantibody of the disclosure. In one example, one or more CDRs from one ormore illustrative human antibodies are combined with CDRs from anantibody from a non-human animal, such as mouse or rat. In anotherexample, all of the CDRs of the chimeric antibody are derived from oneor more illustrative antibodies. In some particular embodiments, thechimeric antibody comprises one, two, or three CDRs from the heavy chainvariable region or from the light chain variable region of anillustrative antibody. Chimeric antibodies can be generated usingconventional methods known in the art.

Another type of modification is to mutate amino acid residues within theCDR1, CDR2 and/or CDR3 regions of the V_(H) and/or V_(L) chain.Site-directed mutagenesis or PCR-mediated mutagenesis can be performedto introduce the mutation(s) and the effect on antibody binding, orother functional property of interest, can be evaluated in in vitro orin vivo assays known in the art. Typically, conservative substitutionsare introduced. The mutations may be amino acid additions and/ordeletions. Moreover, typically no more than one, two, three, four orfive residues within a CDR region are altered. In some embodiments, theantibody derivative comprises 1, 2, 3, or 4 amino acid substitutions inthe H-CDRs and/or in the light chain CDRs. In another embodiment, theamino acid substitution is to change one or more cysteines in anantibody to another residue, such as, without limitation, alanine orserine. The cysteine may be a canonical or non-canonical cysteine. Inone embodiment, the antibody derivative has 1, 2, 3, or 4 conservativeamino acid substitutions in the H-CDR regions relative to the amino acidsequences of an illustrative antibody.

Modifications may also be made to the framework residues within theV_(H) and/or V_(L) regions. Typically, such framework variants are madeto decrease the immunogenicity of the antibody. One approach is to“backmutate” one or more framework residues to the correspondinggermline sequence. An antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis. Several of the illustrativeantibodies of the present disclosure underwent such “back-mutations” tocertain germline sequences, as described further in Example 6.

In addition, modifications may also be made within the Fc region of anillustrative antibody, typically to alter one or more functionalproperties of the antibody, such as serum half-life, complementfixation, Fc receptor binding, and/or antigen-dependent cellularcytotoxicity. In one example, the hinge region of CH1 is modified suchthat the number of cysteine residues in the hinge region is altered,e.g., increased or decreased. This approach is described further in U.S.Pat. No. 5,677,425. The number of cysteine residues in the hinge regionof CH1 is altered to, for example, facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.In another case, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody.

Furthermore, an antibody of the disclosure may be modified to alter itspotential glycosylation site or pattern. Both illustrative antibodiesMOR-7480 and MOR-7483, and any germlined variants thereof, andantibodies that comprise the amino acid sequences of the heavy chainvariable region of MOR-7480 and MOR-7483, comprise a potential N-linkedglycosylation site (NYS) at asparagine 59 in the heavy chain variabledomain. IgG versions of these antibodies further comprise a secondN-linked glycosylation site in the heavy chain Fc domain. Morespecifically, for the IgG2 version of these antibodies, the Fc N-linkedglycosylation site (NST) occurs at asparagine 292 in the heavy chain CH2domain. Thus, each heavy chain can comprise 0-, 1-(at either Fab or Fc)or 2-glycan occupancy such that an antibody comprising two heavy and twolight chains can comprise any combination ranging from 0-glycanoccupancy (i.e., no glycosylation at any of four potential glycosylationsites) to 4-glycan occupancy (i.e., glycosylated at both Fab and Fcsites in each chain). In another aspect, the present disclosure providean derivative of an 4-1BB antibody of the disclosure that contains atleast one mutation in an variable region of a light chain or heavy chainthat changes the pattern of glycosylation in the variable region. Suchan antibody derivative may have an increased affinity and/or a modifiedspecificity for binding an antigen. The mutations may add a novelglycosylation site in the V region, change the location of one or more Vregion glycosylation site(s), or remove a pre-existing V regionglycosylation site. In one embodiment, the present disclosure provides aderivative of an 4-1BB antibody having a potential N-linkedglycosylation site at asparagine 59 in the heavy chain variable region,wherein the potential N-linked glycosylation site in one heavy chainvariable region is removed. In another embodiment, the presentdisclosure provides a derivative of a 4-1BB antibody having a potentialN-linked glycosylation site at asparagine 59 in the heavy chain variableregion, wherein the potential N-linked glycosylation site in both heavychain variable regions is removed. Method of altering the glycosylationpattern of an antibody is known in the art, such as those described inU.S. Pat. No. 6,933,368, the disclosure of which incorporated herein byreference.

In another aspect, the present disclosure provides an antibodyderivative that comprises a 4-1BB antibody, or antigen-binding fragmentthereof, as described herein, linked to an additional molecular entity.Examples of additional molecular entities include pharmaceutical agents,peptides or proteins, detection agent or labels, and antibodies.

In some embodiments, the antibody derivative comprises an antibody ofthe disclosure linked to a pharmaceutical agent. Examples ofpharmaceutical agents include cytotoxic agents or other cancertherapeutic agents, and radioactive isotopes. Specific examples ofcytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents alsoinclude, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Methods for linking an antibody to apharmaceutical agent are known in the art, such as using various linkertechnologies. Examples of linker types include hydrazones, thioethers,esters, disulfides and peptide-containing linkers. For furtherdiscussion of linkers and methods for linking therapeutic agents toantibodies, see also Saito et al., Adv. Drug Deliv. Rev. 55:199-215(2003); Trail, et al., Cancer Immunol. Immunother. 52:328-337 (2003);Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763(2002); Pastan, I. and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091(2002); Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev.53:247-264.

In a particular embodiment, the antibody derivative is a 4-1BB antibodymultimer, which is a multimeric form of a 4-1BB antibody, such asantibody dimers, trimers, or higher-order multimers of monomericantibodies. Individual monomers within an antibody multimer may beidentical or different. In addition, individual antibodies within amultimer may have the same or different binding specificities.Multimerization of antibodies may be accomplished through naturalaggregation of antibodies. For example, some percentage of purifiedantibody preparations (e.g., purified IgG1 molecules) spontaneously formprotein aggregates containing antibody homodimers, and otherhigher-order antibody multimers. Alternatively, antibody homodimers maybe formed through chemical linkage techniques known in the art, such asthrough using crosslinking agents. Suitable crosslinkers include thosethat are heterobifunctional, having two distinctly reactive groupsseparated by an appropriate spacer (such asm-maleimidobenzoyl-N-hydroxysuccinimide ester, succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate, and N-succinimidylS-acethylthio-acetate) or homobifunctional (such as disuccinimidylsuberate). Such linkers are commercially available from, for example,Pierce Chemical Company, Rockford, Ill. Antibodies can also be made tomultimerize through recombinant DNA techniques known in the art.

Examples of other antibody derivatives provided by the presentdisclosure include single chain antibodies, diabodies, domainantibodies, nanobodies, and unibodies. A “single-chain antibody” (scFv)consists of a single polypeptide chain comprising a V_(L) domain linkedto a V_(H) domain wherein V_(L) domain and V_(H) domain are paired toform a monovalent molecule. Single chain antibody can be preparedaccording to method known in the art (see, for example, Bird et al.,(1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883). A “diabody” consists of two chains, each chaincomprising a heavy chain variable region connected to a light chainvariable region on the same polypeptide chain connected by a shortpeptide linker, wherein the two regions on the same chain do not pairwith each other but with complementary domains on the other chain toform a bispecific molecule. Methods of preparing diabodies are known inthe art (See, e.g., Holliger P. et al., (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448, and Poljak R. J. et al., (1994) Structure2:1121-1123). Domain antibodies (dAbs) are small functional bindingunits of antibodies, corresponding to the variable regions of either theheavy or light chains of antibodies. Domain antibodies are wellexpressed in bacterial, yeast, and mammalian cell systems. Furtherdetails of domain antibodies and methods of production thereof are knownin the art (see, for example, U.S. Pat. Nos. 6,291,158; 6,582,915;6,593,081; 6,172,197; 6,696,245; European Patents 0368684 & 0616640;WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019 andWO03/002609). Nanobodies are derived from the heavy chains of anantibody. A nanobody typically comprises a single variable domain andtwo constant domains (CH2 and CH3) and retains antigen-binding capacityof the original antibody. Nanobodies can be prepared by methods known inthe art (See e.g., U.S. Pat. No. 6,765,087, U.S. Pat. No. 6,838,254, WO06/079372). Unibodies consist of one light chain and one heavy chain ofa IgG4 antibody. Unibodies may be made by the removal of the hingeregion of IgG4 antibodies. Further details of unibodies and methods ofpreparing them may be found in WO2007/059782.

C. Nucleic Acids, Vectors, Host Cells, and Recombinant Methods ofProducing 4-1BB Antibodies

Another aspect of the disclosure provides an isolated nucleic acidmolecule that comprises a nucleotide sequence encoding an amino acidsequence of a binding molecule provided by the present disclosure. Theamino acid sequence encoded by the nucleotide sequence may be anyportion of an antibody, such as a CDR, a sequence comprising one, two,or three CDRs, a variable region of a heavy chain, variable region of alight chain, or may be a full-length heavy chain or full length lightchain. A nucleic acid of the disclosure can be, for example, DNA or RNA,and may or may not contain intronic sequences. Typically, the nucleicacid is a cDNA molecule.

In some embodiments, the disclosure provides an isolated nucleic acidmolecule that comprises or consists of a nucleotide sequence encoding anamino acid sequence selected from the group consisting of: (1) aminoacid sequence of a H-CDR3 or L-CRD3 of an illustrative antibody; (2) avariable region of a heavy chain or variable region of a light chain ofan illustrative antibody; or (3) a full length heavy chain or fulllength light chain of an illustrative antibody.

In other embodiments, the nucleic acid molecule comprises or consists ofa nucleotide sequence that encodes an amino acid sequence as set forthin any one of SEQ ID NOs:1-10, 15-24, 29-38, 43, 44, 45, 46, 51, 52,55-57, 60, 61, 64, and 65.

In still other embodiments, the nucleic acid molecule comprises orconsists of nucleotide sequence selected from the group consisting ofSEQ ID NOs: 11-14, 25-28, 39-42, 47-50, 53, 54, 58, 59, 62, 63, 66, and67.

Nucleic acids of the disclosure can be obtained using any suitablemolecular biology techniques. For antibodies expressed by hybridomas,cDNAs encoding the light and heavy chains of the antibody made by thehybridoma can be obtained by PCR amplification or cDNA cloningtechniques. For antibodies obtained from an immunoglobulin gene library(e.g., using phage display techniques), the nucleic acid encoding theantibody can be recovered from the library.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. The IgG1 constant region sequence canbe any of the various alleles or allotypes known to occur amongdifferent individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). Theseallotypes represent naturally occurring amino acid substitution in theIgG1 constant regions. For a Fab fragment heavy chain gene, theV_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabatet al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature348:552-554 (1990)).

The present disclosure further provides a vector that comprises anucleic acid molecule provided by the present disclosure. The nucleicacid molecule may encode a portion of a light chain or heavy chain (suchas a CDR or a variable region), a full-length light or heavy chain,polypeptide that comprises a portion or full-length of a heavy or lightchain, or an amino acid sequence of an antibody derivative orantigen-binding fragment. In some embodiments, the vector is anexpression vector useful for the expression of a binding molecule, suchas an antibody or an antigen binding fragment thereof.

To express a binding molecule of the disclosure, DNAs encoding partialor full-length light and heavy chains are inserted into expressionvectors such that the DNA molecules are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” means that an antibody gene is ligatedinto a vector such that transcriptional and translational controlsequences within the vector serve their intended function of regulatingthe transcription and translation of the DNA molecule. The expressionvector and expression control sequences are chosen to be compatible withthe expression host cell used. The antibody light chain gene and theantibody heavy chain gene can be inserted into separate vector or, moretypically, both genes are inserted into the same expression vector. Theantibody genes are inserted into the expression vector by any suitablemethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype and subclass byinserting them into expression vectors already encoding heavy chainconstant and light chain constant regions of the desired isotype andsubclass such that the V_(H) segment is operatively linked to the C_(H)segment(s) within the vector and the V_(K) segment is operatively linkedto the C_(L) segment within the vector. Additionally or alternatively,the recombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the expression vectors of thedisclosure typically carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Examples of regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SR promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, theexpression vectors may carry additional sequences, such as sequencesthat regulate replication of the vector in host cells (e.g., origins ofreplication) and selectable marker genes. The selectable marker genefacilitates selection of host cells into which the vector has beenintroduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Selectable marker genes include the dihydrofolate reductase (DHFR) gene(for use in dhfr-host cells with methotrexate selection/amplification)and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell byany suitable techniques. The various forms of the term “transfection”are intended to encompass a wide variety of techniques commonly used forthe introduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is possible toexpress the antibodies of the disclosure in either prokaryotic oreukaryotic host cells, expression of antibodies in eukaryotic cells, andtypically mammalian host cells, is most typical.

The present disclosure further provides a host cell containing a nucleicacid molecule provided by the present disclosure. The host cell can bevirtually any cell for which expression vectors are available. It maybe, for example, a higher eukaryotic host cell, such as a mammaliancell, a lower eukaryotic host cell, such as a yeast cell, and may be aprokaryotic cell, such as a bacterial cell. Introduction of therecombinant nucleic acid construct into the host cell can be effected bycalcium phosphate transfection, DEAE, dextran mediated transfection,electroporation or phage infection.

Suitable prokaryotic hosts for transformation include E. coli, Bacillussubtilis, Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus.

Mammalian host cells for expressing a binding molecule of the disclosureinclude, for example, Chinese Hamster Ovary (CHO) cells (includingdhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci.USA 77:4216-4220 (1980), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J. Mol. Biol. 159:601-621 (1982), NS0myeloma cells, COS cells and Sp2 cells. In particular, for use with NS0myeloma or CHO cells, another expression system is the GS (glutaminesynthetase) gene expression system disclosed in WO 87/04462, WO 89/01036and EP 338,841. When expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using any suitableprotein purification methods.

D. Compositions

In other aspects, the present disclosure provides a compositioncontaining a binding molecule provided by the disclosure. In one aspect,the composition is a pharmaceutical composition comprising a bindingmolecule and a pharmaceutically acceptable carrier. The compositions canbe prepared by conventional methods known in the art.

In some embodiments, present disclosure provides a compositioncomprising an antibody, or an antigen-binding portion thereof, providedby the present disclosure and a pharmaceutically acceptable carrier,wherein said antibody comprises a variable domain comprising the CDRamino acid sequence set forth in SEQ ID NO:30, and wherein saidcomposition comprises not more than about 11%, 10%, 8%, 5%, 3%, or 2% ofsaid antibody, or antigen-binding portion, that is glycosylated at theasparagine of said amino acid sequence compared with the total amount ofantibody, or antigen-binding portion thereof, present in saidcomposition. In another embodiment, the composition comprises at leastabout 2% of said antibody, or antigen-binding portion, that isglycosylated at the asparagine of said amino acid sequence of SEQ IDNO:30 compared with the total amount of antibody, or antigen-bindingportion thereof, present in said composition.

The term “pharmaceutically acceptable carrier” refers to any inactivesubstance that is suitable for use in a formulation for the delivery ofa binding molecule. A carrier may be an antiadherent, binder, coating,disintegrant, filler or diluent, preservative (such as antioxidant,antibacterial, or antifungal agent), sweetener, absorption delayingagent, wetting agent, emulsifying agent, buffer, and the like. Examplesof suitable pharmaceutically acceptable carriers include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like) dextrose, vegetable oils (such as olive oil), saline, buffer,buffered saline, and isotonic agents such as sugars, polyalcohols,sorbitol, and sodium chloride.

The compositions may be in any suitable forms, such as liquid,semi-solid, and solid dosage forms. Examples of liquid dosage formsinclude solution (e.g., injectable and infusible solutions),microemulsion, liposome, dispersion, or suspension. Examples of soliddosage forms include tablet, pill, capsule, microcapsule, and powder. Aparticular form of the composition suitable for delivering a bindingmolecule is a sterile liquid, such as a solution, suspension, ordispersion, for injection or infusion. Sterile solutions can be preparedby incorporating the antibody in the required amount in an appropriatecarrier, followed by sterilization microfiltration. Generally,dispersions are prepared by incorporating the antibody into a sterilevehicle that contains a basic dispersion medium and other carriers. Inthe case of sterile powders for the preparation of sterile liquid,methods of preparation include vacuum drying and freeze-drying(lyophilization) to yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The various dosage forms of the compositions can beprepared by conventional techniques known in the art.

The relative amount of a binding molecule included in the compositionwill vary depending upon a number of factors, such as the specificbinding molecule and carriers used, dosage form, and desired release andpharmacodynamic characteristics. The amount of a binding molecule in asingle dosage form will generally be that amount which produces atherapeutic effect, but may also be a lesser amount. Generally, thisamount will range from about 0.01 percent to about 99 percent, fromabout 0.1 percent to about 70 percent, or from about 1 percent to about30 percent relative to the total weight of the dosage form.

In addition to the binding molecule, one or more additional therapeuticagents may be included in the composition. Examples of additionaltherapeutic agents are described herein below. The suitable amount ofthe additional therapeutic agent to be included in the composition canbe readily selected by a person skilled in the art, and will varydepending on a number of factors, such as the particular agent andcarriers used, dosage form, and desired release and pharmacodynamiccharacteristics. The amount of the additional therapeutic agent includedin a single dosage form will generally be that amount of the agent whichproduces a therapeutic effect, but may be a lesser amount as well.

E. Use of the Binding Molecules and Pharmaceutical Compositions

Binding molecules and pharmaceutical compositions provided by thepresent disclosure are useful for therapeutic, diagnostic, or otherpurposes, such as enhancing an immune response, treating cancer,enhancing efficacy of other cancer therapy, enhancing vaccine efficacy,or treating autoimmune diseases. Thus, in other aspects, the presentdisclosure provides methods of using the binding molecules orpharmaceutical compositions. In one aspect, the present disclosureprovides a method of treating a disorder in a mammal, which comprisesadministering to the mammal in need of treatment a therapeuticallyeffective amount of a binding molecule provided by the disclosure. Thebinding molecule may be a 4-1BB agonist or antagonist. In someembodiments, the binding molecule is a 4-1BB agonist. In someembodiments, the mammal is a human.

In some embodiments, the disorder is a cancer. A variety of cancerswhere 4-1BB is implicated, whether malignant or benign and whetherprimary or secondary, may be treated or prevented with a method providedby the disclosure. Examples of such cancers include lung cancers such asbronchogenic carcinoma (e.g., squamous cell carcinoma, small cellcarcinoma, large cell carcinoma, and adenocarcinoma), alveolar cellcarcinoma, bronchial adenoma, chondromatous hamartoma (noncancerous),and sarcoma (cancerous); heart cancer such as myxoma, fibromas, andrhabdomyomas; bone cancers such as osteochondromas, condromas,chondroblastomas, chondromyxoid fibromas, osteoid osteomas, giant celltumors, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcomas,malignant fibrous histiocytomas, Ewing's tumor (Ewing's sarcoma), andreticulum cell sarcoma; brain cancer such as gliomas (e.g., glioblastomamultiforme), anaplastic astrocytomas, astrocytomas, oligodendrogliomas,medulloblastomas, chordoma, Schwannomas, ependymomas, meningiomas,pituitary adenoma, pinealoma, osteomas, hemangioblastomas,craniopharyngiomas, chordomas, germinomas, teratomas, dermoid cysts, andangiomas; cancers in digestive system such as leiomyoma, epidermoidcarcinoma, adenocarcinoma, leiomyosarcoma, stomach adenocarcinomas,intestinal lipomas, intestinal neurofibromas, intestinal fibromas,polyps in large intestine, and colorectal cancers; liver cancers such ashepatocellular adenomas, hemangioma, hepatocellular carcinoma,fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, andangiosarcoma; kidney cancers such as kidney adenocarcinoma, renal cellcarcinoma, hypernephroma, and transitional cell carcinoma of the renalpelvis; bladder cancers; hematological cancers such as acute lymphocytic(lymphoblastic) leukemia, acute myeloid (myelocytic, myelogenous,myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia(e.g., Sezary syndrome and hairy cell leukemia), chronic myelocytic(myeloid, myelogenous, granulocytic) leukemia, Hodgkin's lymphoma,non-Hodgkin's lymphoma, B cell lymphoma, mycosis fungoides, andmyeloproliferative disorders (including myeloproliferative disorderssuch as polycythemia vera, myelofibrosis, thrombocythemia, and chronicmyelocytic leukemia); skin cancers such as basal cell carcinoma,squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget'sdisease; head and neck cancers; eye-related cancers such asretinoblastoma and intraoccular melanocarcinoma; male reproductivesystem cancers such as benign prostatic hyperplasia, prostate cancer,and testicular cancers (e.g., seminoma, teratoma, embryonal carcinoma,and choriocarcinoma); breast cancer; female reproductive system cancerssuch as uterine cancer (endometrial carcinoma), cervical cancer(cervical carcinoma), cancer of the ovaries (ovarian carcinoma), vulvarcarcinoma, vaginal carcinoma, fallopian tube cancer, and hydatidiformmole; thyroid cancer (including papillary, follicular, anaplastic, ormedullary cancer); pheochromocytomas (adrenal gland); noncancerousgrowths of the parathyroid glands; pancreatic cancers; and hematologicalcancers such as leukemias, myelomas, non-Hodgkin's lymphomas, andHodgkin's lymphomas.

In some other embodiments, the disorder is an autoimmune disease.Examples of autoimmune diseases that may be treated with the bindingmolecules include autoimmune encephalomyelitis, lupus erythematosus, andrheumatoid arthritis. The binding molecule may also be used to treatinflammation (such as allergic asthma) and chronic graft-versus-hostdisease,

In another aspect, the present disclosure provides a method of enhancingan immune response in a mammal, which comprises administering to themammal a therapeutically effective amount of a binding molecule providedby the disclosure. In some embodiments, the binding molecule is a 4-1BBantibody or antigen-binding fragment thereof and the mammal is a human.In a further embodiment, the binding molecule is 4-1BB agonist antibodyor an antigen-binding fragment thereof. The term “enhancing immuneresponse” or its grammatical variations, means stimulating, evoking,increasing, improving, or augmenting any response of a mammal's immunesystem. The immune response may be a cellular response (i.e.cell-mediated, such as cytotoxic T lymphocyte mediated) or a humoralresponse (i.e. antibody mediated response), and may be a primary orsecondary immune response. Examples of enhancement of immune responseinclude increased CD4+ helper T cell activity and generation ofcytolytic T cells. The enhancement of immune response can be assessedusing a number of in vitro or in vivo measurements known to thoseskilled in the art, including, but not limited to, cytotoxic Tlymphocyte assays, release of cytokines (for example IL-2 production),regression of tumors, survival of tumor bearing animals, antibodyproduction, immune cell proliferation, expression of cell surfacemarkers, and cytotoxicity. Typically, methods of the disclosure enhancethe immune response by a mammal when compared to the immune response byan untreated mammal or a mammal not treated using the claimed methods.In one embodiment, the binding molecule is used to enhance the immuneresponse of a human to a microbial pathogen (such as a virus). Inanother embodiment, the binding molecule is used to enhance the immuneresponse of a human to a vaccine. The binding molecule may be a 4-1BBagonist or antagonist. In some embodiments, the binding molecule is a4-1BB agonist. In one embodiment, the method enhances a cellular immuneresponse, particularly a cytotoxic T cell response. In anotherembodiment, the cellular immune response is a T helper cell response. Instill another embodiment, the immune response is a cytokine production,particularly IL-2 production. The binding molecule may be used toenhance the immune response of a human to a microbial pathogen (such asa virus) or to a vaccine. The binding molecule may be a 4-1BB agonist orantagonist. In some embodiments, the binding molecule is a 4-1BBagonist.

In practicing the therapeutic methods, the binding molecules may beadministered alone as monotherapy, or administered in combination withone or more additional therapeutic agents or therapies. Thus, in anotheraspect, the present disclosure provides a combination therapy, whichcomprises a binding molecule in combination with one or more additionaltherapies or therapeutic agents for separate, sequential or simultaneousadministration. The term “additional therapy” refers to a therapy whichdoes not employ a binding molecule provided by the disclosure as atherapeutic agent. The term “additional therapeutic agent” refers to anytherapeutic agent other than a binding molecule provided by thedisclosure. In one particular aspect, the present disclosure provides acombination therapy for treating cancer in a mammal, which comprisesadministering to the mammal a therapeutically effective amount of abinding molecule provided by the disclosure in combination with one ormore additional therapeutic agents. In a further embodiment, the mammalis a human.

A wide variety of cancer therapeutic agents may be used in combinationwith a binding molecule provided by the present disclosure. One ofordinary skill in the art will recognize the presence and development ofother cancer therapies which can be used in combination with the methodsand binding molecules of the present disclosure, and will not berestricted to those forms of therapy set forth herein. Examples ofcategories of additional therapeutic agents that may be used in thecombination therapy for treating cancer include (1) chemotherapeuticagents, (2) immunotherapeutic agents, and (3) hormone therapeuticagents.

The term “chemotherapeutic agent” refers to a chemical or biologicalsubstance that can cause death of cancer cells, or interfere withgrowth, division, repair, and/or function of cancer cells. Examples ofchemotherapeutic agents include those that are disclosed in WO2006/129163, and US 20060153808, the disclosures of which areincorporated herein by reference. Examples of particularchemotherapeutic agents include: (1) alkylating agents, such aschlorambucil (LEUKERAN), mcyclophosphamide (CYTOXAN), ifosfamide (IFEX),mechlorethamine hydrochloride (MUSTARGEN), thiotepa (THIOPLEX),streptozotocin (ZANOSAR), carmustine (BICNU, GLIADEL WAFER), lomustine(CEENU), and dacarbazine (DTIC-DOME); (2) alkaloids or plant vincaalkaloids, including cytotoxic antibiotics, such as doxorubicin(ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin(CERUBIDINE, DAUNOXOME), nemorubicin, idarubicin (IDAMYCIN PFS,ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE). dactinomycin (actinomycin D,COSMEGEN), plicamycin (MITHRACIN), mitomycin (MUTAMYCIN), and bleomycin(BLENOXANE), vinorelbine tartrate (NAVELBINE)), vinblastine (VELBAN),vincristine (ONCOVIN), and vindesine (ELDISINE); (3) antimetabolites,such as capecitabine (XELODA), cytarabine (CYTOSAR-U), fludarabine(FLUDARA), gemcitabine (GEMZAR), hydroxyurea (HYDRA), methotrexate(FOLEX, MEXATE, TREXALL), nelarabine (ARRANON), trimetrexate(NEUTREXIN), and pemetrexed (ALIMTA); (4) Pyrimidine antagonists, suchas 5-fluorouracil (5-FU); capecitabine (XELODA), raltitrexed (TOMUDEX),tegafur-uracil (UFTORAL), and gemcitabine (GEMZAR); (5) taxanes, such asdocetaxel (TAXOTERE), paclitaxel (TAXOL); (6) platinum drugs, such ascisplatin (PLATINOL) and carboplatin (PARAPLATIN), and oxaliplatin(ELOXATIN); (7) topoisomerase inhibitors, such as irinotecan(CAMPTOSAR), topotecan (HYCAMTIN), etoposide (ETOPOPHOS, VEPESSID,TOPOSAR), and teniposide (VUMON); (8) epipodophyllotoxins(podophyllotoxin derivatives), such as etoposide (ETOPOPHOS, VEPESSID,TOPOSAR); (9) folic acid derivatives, such as leucovorin (WELLCOVORIN);(10) nitrosoureas, such as carmustine (BiCNU), lomustine (CeeNU); (11)inhibitors of receptor tyrosine kinase, including epidermal growthfactor receptor (EGFR), vascular endothelial growth factor (VEGF),insulin receptor, insulin-like growth factor receptor (IGFR), hepatocytegrowth factor receptor (HGFR), and platelet-derived growth factorreceptor (PDGFR), such as gefitinib (IRESSA), erlotinib (TARCEVA),bortezomib (VELCADE), imatinib mesylate (GLEEVEC), genefitinib,lapatinib, sorafenib, thalidomide, sunitinib (SUTENT), axitinib,rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), cetuximab(ERBITUX), bevacizumab (AVASTIN), and ranibizumab (LUCENTIS), lym-1(ONCOLYM), antibodies to insulin-like growth factor-1 receptor (IGF-1R)that are disclosed in WO2002/053596); (12) angiogenesis inhibitors, suchas bevacizumab (AVASTIN), suramin (GERMANIN), angiostatin, SU5416,thalidomide, and matrix metalloproteinase inhibitors (such as batimastatand marimastat), and those that are disclosed in WO2002055106; and (13)proteasome inhibitors, such as bortezomib (VELCADE).

The term “immunotherapeutic agents” refers to a chemical or biologicalsubstance that can enhance an immune response of a mammal. Examples ofimmunotherapeutic agents include: bacillus Calmette-Guerin (BCG);cytokines such as interferons; vaccines such as MyVax personalizedimmunotherapy, Onyvax-P, Oncophage, GRNVAC1, FavId, Provenge, GVAX,Lovaxin C, BiovaxiD, GMXX, and NeuVax; and antibodies such asalemtuzumab (CAMPATH), bevacizumab (AVASTIN), cetuximab (ERBITUX),gemtuzunab ozogamicin (MYLOTARG), ibritumomab tiuxetan (ZEVALIN),panitumumab (VECTIBIX), rituximab (RITUXAN, MABTHERA), trastuzumab(HERCEPTIN), tositumomab (BEXXAR), ipilimumab (YERVOY) tremelimumab,CAT-3888, agonist antibodies to OX40 receptor (such as those disclosedin WO2009/079335), agonist antibodies to CD40 receptor (such as thosedisclosed in WO2003/040170, and TLR-9 agonists (such as those disclosedin WO2003/015711, WO2004/016805, and WO2009/022215).

The term “hormone therapeutic agent” refers to a chemical or biologicalsubstance that inhibits or eliminates the production of a hormone, orinhibits or counteracts the effect of a hormone on the growth and/orsurvival of cancerous cells. Examples of such agents suitable for themethods herein include those that are disclosed in US20070117809.Examples of particular hormone therapeutic agents include tamoxifen(NOLVADEX), toremifene (Fareston), fulvestrant (FASLODEX), anastrozole(ARIMIDEX), exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate(MEGACE), goserelin (ZOLADEX), and leuprolide (LUPRON). The bindingmolecules of this disclosure may also be used in combination withnon-drug hormone therapies such as (1) surgical methods that remove allor part of the organs or glands which participate in the production ofthe hormone, such as the ovaries, the testicles, the adrenal gland, andthe pituitary gland, and (2) radiation treatment, in which the organs orglands of the patient are subjected to radiation in an amount sufficientto inhibit or eliminate the production of the targeted hormone.

The combination therapy for treating cancer also encompasses thecombination of a binding molecule with surgery to remove a tumor. Thebinding molecule may be administered to the mammal before, during, orafter the surgery.

The combination therapy for treating cancer also encompasses combinationof a binding molecule with radiation therapy, such as ionizing(electromagnetic) radiotherapy (e.g., X-rays or gamma rays) and particlebeam radiation therapy (e.g., high linear energy radiation). The sourceof radiation can be external or internal to the mammal. The bindingmolecule may be administered to the mammal before, during, or after theradiation therapy.

The binding molecules and compositions provided by the presentdisclosure can be administered via any suitable enteral route orparenteral route of administration. The term “enteral route” ofadministration refers to the administration via any part of thegastrointestinal tract. Examples of enteral routes include oral,mucosal, buccal, and rectal route, or intragastric route. “Parenteralroute” of administration refers to a route of administration other thanenteral route. Examples of parenteral routes of administration includeintravenous, intramuscular, intradermal, intraperitoneal, intratumor,intravesical, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, transtracheal, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal, subcutaneous, or topicaladministration. The antibodies and compositions of the disclosure can beadministered using any suitable method, such as by oral ingestion,nasogastric tube, gastrostomy tube, injection, infusion, implantableinfusion pump, and osmotic pump. The suitable route and method ofadministration may vary depending on a number of factors such as thespecific antibody being used, the rate of absorption desired, specificformulation or dosage form used, type or severity of the disorder beingtreated, the specific site of action, and conditions of the patient, andcan be readily selected by a person skilled in the art

The term “therapeutically effective amount” of a binding molecule refersto an amount that is effective for an intended therapeutic purpose. Forexample, in the context of enhancing an immune response, a“therapeutically effective amount” is any amount that is effective instimulating, evoking, increasing, improving, or augmenting any responseof a mammal's immune system. In the context of treating a disease, a“therapeutically effective amount” is any amount that is sufficient tocause any desirable or beneficial effect in the mammal being treated.Specifically, in the treatment of cancer, examples of desirable orbeneficial effects include inhibition of further growth or spread ofcancer cells, death of cancer cells, inhibition of reoccurrence ofcancer, reduction of pain associated with the cancer, or improvedsurvival of the mammal. The therapeutically effective amount of a 4-1BBantibody usually ranges from about 0.001 to about 500 mg/kg, and moreusually about 0.01 to about 100 mg/kg, of the body weight of the mammal.For example, the amount can be about 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5mg/kg, 10 mg/kg, 50 mg/kg, or 100 mg/kg of body weight of the mammal. Insome embodiments, the therapeutically effective amount of a 4-1BBantibody is in the range of about 0.01-30 mg/kg of body weight of themammal. In some other embodiments, the therapeutically effective amountof a 4-1BB antibody is in the range of about 0.05-15 mg/kg of bodyweight of the mammal. The precise dosage level to be administered can bereadily determined by a person skilled in the art and will depend on anumber of factors, such as the type, and severity of the disorder to betreated, the particular binding molecule employed, the route ofadministration, the time of administration, the duration of thetreatment, the particular additional therapy employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A binding molecule or composition is usually administered on multipleoccasions. Intervals between single doses can be, for example, weekly,monthly, every three months or yearly. An exemplary treatment regimenentails administration once per week, once every two weeks, once everythree weeks, once every four weeks, once a month, once every threemonths or once every three to six months. Typical dosage regimens for a4-1BB antibody include 1 mg/kg body weight or 3 mg/kg body weight viaintravenous administration, using one of the following dosing schedules:(i) every four weeks for six dosages, then every three months; (ii)every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kgbody weight every three weeks.

The present disclosure is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this disclosure are expressly incorporated herein byreference in their entirety.

EXAMPLES Example 1 Generation of Fab Fragments that Bind to 4-1BB

Certain antibodies provided by the present invention were originallygenerated from Fabs that bind to human 4-1BB. The Fabs were selectedfrom a phage display library, the MorphoSys HuCAL GOLD® phagemidlibrary, following alternating panning on 4-1BB FC and cells expressinghuman 4-1BB. These Fabs include those that are designated as “Fab-6032,”“Fab-7361,” “Fab-7480,” and “Fab-7483.” 4-1BB antibodies MOR-6032,MOR-7361, MOR-7480, and MOR-7483 disclosed in this application weregenerated from “Fab-6032,” “Fab-7361,” “Fab-7480,” and “Fab-7483,respectively. The amino acid sequence of the light chain variable regionand heavy chain variable region of a given Fab are identical to theamino acid sequence of the light chain variable region and heavy chainvariable region, respectively, of an illustrative antibody thedesignation of which shares the same numerical number with thedesignation of the Fab. For example, Fab-7480 and antibody MOR-7480 haveidentical amino acid sequences for their light chain variable region andheavy chain variable region, respectively.

The phagemid library is based on the HuCAL® concept (Knappik et al.,2000, J. Mol. Biol. 296(1):57-86) and employs the CysDisplay™ technologyfor displaying the Fab on the phage surface (Löhning, WO 01/05950).HuCAL GOLD® provides the option of performing selections with six singlesub-libraries each comprising one VH (VH1, VH2, VH3, VH4, VH5, VH6)master gene combined with all seven VL master genes or performingselections using combined phage pools. Phage for the 1st round ofpannings were prepared by Hyperphage (M13KO07ΔpIII, obtained fromProgen, Heidelberg, Germany). HuCAL GOLD® is described in detail inChristine Rothe, et. al, J. Mol. Biol. (2008) 376, 1182-1200.

Solid phase panning was performed using recombinant human 4-1BB-Fc (R&DSystems, Cat. No. 838-4B; Minneapolis, Minn.).

Example 2 Characterizations of FABS

The characterizations of the four Fabs described in example 1 weredetermined in the assays described below using the monovalent Fab-formatcomprising a Fab having a Flag/His-Tag.

2A. Affinity Determined with Solution Equilibrium Titration (SET) Method

The affinity (as expressed as K_(D)) of the four Fabs was determinedusing the SET method using an instrumentation from Meso Scale Discovery(“MSD”). Monomer fractions of antibody protein were used (at least 90%monomer content, analyzed by analytical SEC; Superdex75 (AmershamPharmacia) for Fab, or Tosoh G3000SWXL (Tosoh Bioscience) for IgG,respectively).

Affinity determination in solution was basically performed as describedin the literature (Friguet et al. 1985). In order to improve thesensitivity and accuracy of the SET method, it was transferred fromclassical ELISA to ECL based technology (Haenel et al. 2005).

1 mg/ml goat-anti-human (Fab)₂ fragment specific antibodies (Dianova)were labeled with MSD Sulfo-TAG™ NHS-Ester (Meso Scale Discovery,Gaithersburg, Md., USA) according to manufacturer's instructions.

The experiments were carried out in polypropylene microtiter plates andPBS pH 7.4 with 0.5% BSA and 0.02% Tween 20 as assay buffer. Unlabeledhuman 4-1BB was diluted in a 2^(n) series, starting with a concentrationat least 10 times higher than the expected K_(D). Wells without antigenwere used to determine B_(max) values; wells with assay buffer were usedto determine background. After addition of, e.g., 30 pM Fab (finalconcentration in 60 μL final volume), the mixture was incubatedovernight at room temperature. The Fab concentration applied was similarto or below the expected K_(D).

Standard MSD plates were coated with 0.05 μg/ml human 4-1BB in PBS (30μL/well), incubated overnight, and blocked with 3% BSA in PBS for 1hour. After washing the plate with assay buffer, the equilibratedsamples were transferred to those plates (30 μL per well) and incubatedfor 20 minutes. After washing, 30 μL/well of the MSD Sulfo-tag labeleddetection antibody (goat anti-human (Fab)₂) in a final dilution of1:1500 was added to the MSD plate and incubated for 30 min on anEppendorf shaker (700 rpm).

After washing the plate and adding 30 μL/well MSD Read Buffer T withsurfactant, electrochemiluminescence signals were detected using aSector Imager 6000 (Meso Scale Discovery, Gaithersburg, Md., USA).

The data were evaluated with XLfit (IDBS) software applying customizedfitting models. For K_(D) determination of Fab molecules the followingfit model was used (Haenel et al., 2005) and modified according toAbraham et al. (1996, J. Molec. Recog. 9(5-6):456-461):

$y = {B_{\max} - \left( {\frac{B_{\max}}{{2\lbrack{Fab}\rbrack}_{t}}\left( {\lbrack{Fab}\rbrack_{t} + x + K_{D} - \sqrt{\left( {\lbrack{Fab}\rbrack_{t} + x + K_{D}} \right)^{2} - {4{x\lbrack{Fab}\rbrack}_{t}}}} \right)} \right)}$

[Fab]_(t): Applied total Fab concentration

x: Applied total soluble antigen concentration (binding sites)

B_(max): Maximal signal of Fab without antigen

K_(D): Affinity

Results are presented in Table 3.

2B. Biacore K_(D) Determination on Directly Coated Antigen

For K_(D) determination, monomeric Fab fractions (at least 90% monomercontent, analyzed by analytical SEC; Superdex75, Amersham Pharmacia)were used as analyte. Binding to immobilized antigen was analyzed usinga BIAcore3000 instrument (Biacore, Sweden).

The kinetic rate constants k_(on) and k_(off) were determined withserial dilutions of the respective Fab binding to covalently immobilizedantigen CD137/HUMAN 4-1BB using the Biacore 3000 instrument (Biacore,Uppsala, Sweden). For covalent antigen immobilization standard EDC-NHSamine coupling chemistry was used. Kinetic measurements were done inHBS-EP (10 mM HEPES; pH 7.4; 150 mM NaCl; 3 mM EDTA; Tween20 0.005%) ata flow rate of 20 μl/minute using Fab concentrations ranging from about16 to 500 nM. The injection time for each concentration was 1 minute,followed by at least 3 minutes dissociation phase. For regeneration, oneor more 5 μl injections of Glycine/HCl pH2 were used.

For K_(D) estimation of whole IgG molecules, IgGs were injected assamples on a F1 sensor chip with a low density of covalently immobilizedhuman 4-1BB (approx. 130 RU) using a 2^(n) serial dilution withconcentrations ranging from 16 to 500 nM. Sensorgrams were evaluatedusing a bivalent fit model a qualitatively compared to rank thecorresponding K_(D) values.

All sensorgrams were fitted using BIA evaluation software 3.1 (Biacore).Results are presented in Table 3.

2C. Binding of Fabs in ELISA Assay

The binding of the four Fabs were determined using standard ELISAtechniques on directly coated human 4-1BB/Fc. Results are presented inTable 3.

2D. Binding of Fabs in FACS Assay

The binding of the four Fabs was determined using standard FACS assaytechniques on HEK293 cells stably transfected and expressing human 4-1BBas well as 300.19 (murine B-cell line) negative control cells. Theresults are presented in Table 3.

TABLE 3 Binding Properties of Fabs BIAcore Affinity SET Affinity ELISAAssay FACS Assay Fab K_(D) [nM] K_(D) [pM] EC50 [nM] EC50 [nM] Fab-603266 Not 1.0 270 measured Fab-7361 118 Not 0.6 105 measured Fab-7480 0.546 0.7 0.9 Fab-7483 0.7 43 0.6 8.9

Example 3 Characterization of IgGs

Several Fabs obtained from the panning as described herein, includingFab-6032, Fab-7361, Fab-7480, and Fab-7483, were selected for conversioninto full length antibodies in IgG1 and IgG4 formats for furthercharacterizations as described in this example. The four illustrativeantibodies identified in this example, ie., MOR-6032, MOR-7361,MOR-7480, and MOR-7483, were converted from Fab-6032, Fab-7361,Fab-7480, and Fab-7483, respectively. The antibodies in IgG format wereexpressed and purified, and then characterized in ELISA, FACS, andluciferase reporter gene assays.

3A. Conversion to IqG

In order to express full length IgG, variable domain fragments of heavy(VH) and light chains (VL) were subcloned from Fab expression vectorsinto appropriate pMorph®_hIgG vectors for human IgG1 and human IgG4.

3B. Transient Expression and Purification of Human IqG

Transient expression of full length human IgG was performed in HKB11cells, which were transfected with IgG heavy and light chain expressionvectors at a 1:1 ratio. Cell culture supernatant was harvested aftertransfection and upscaled to 3-fold transfection volume, respectively.Supernatant was cleared by centrifugation and filtration, and thensubjected to standard protein A affinity chromatography (MabSelect SURE,GE Healthcare). Proteins were eluted and neutralized. Further downstreamprocessing involved buffer exchange and sterile filtration. Proteinconcentrations were determined by UV-spectrophotometry. Purity of IgGwas analyzed under denaturing, reducing and denaturing, non-reducingconditions in SDS-PAGE or by using Agilent BioAnalyzer. HP-SEC wasperformed to analyze IgG preparations in native state.

3C. Characterization of IgGs in ELISA Assay

IgGs were used for ELISA binding characterization on human 4-1BB/Fc andmouse 4-1BB/Fc in a direct coated setup. Table 4 below sets out theELISA binding results for antibodies MOR-6032, MOR-7361, MOR-7480, andMOR-7483, all in IgG1 format.

TABLE 4 Binding of IgG1s in ELISA Assay Human 4-1BB/Fc Antibody EC₅₀[nM] Mouse 4-1BB/Fc MOR-6032 0.2 − MOR-7361 0.3 − MOR-7480 0.7 (+)MOR-7483 0.9 −

3D. Binding Selectivity of Antibodies (FACS Assay)

The selectivity of antibodies for 4-1BB was assessed againstextracellular domain protein of 4-1BB and other members of the TNFRsuperfamily. These receptors included CD40 (TNFRSF5) and OX-40 (CD134,TNFRSF4). IgGs were used for FACS binding characterization on negativecontrol HEK293 cells, as well as HEK293T-h4-1BB cells stably transfectedand expressing human 4-1BB, and 300.19 stably transfected cellsexpressing OX-40, and 300.19 cells stably transfected and expressingCD40. The FACS binding results for antibodies MOR-6032, MOR-7361,MOR-7480, and MOR-7483, all in IgG1 format, are presented in Table 5. Nosignificant binding to OX-40 or CD40 was observed at concentrations upto 1000 nM, demonstrating that the antibodies are at least 100-fold moreselective for 4-1BB versus other related family members tested.

TABLE 5 Binding Selectivity of Antibodies (IgG1) in FACS Assay HEK293T4-1BB parental 300.19 300.19 300.19 Antibody EC₅₀ [nM] HEK293 OX-40 CD40parental MOR-6032 0.6 − − − − MOR-7361 0.8 − − − − MOR-7480 0.6 − − − −MOR-7483 0.5 (+) − − −

3E. Characterization of IqGs in Luciferase Reporter Gene Assay

IgGs were also characterized for binding in a luciferase reporter geneassay using HEK293T-h4-1BB cells in a plate bound assay, a solublebinding assay, and cross-linked binding assay. Table 6 sets out theresults of the luciferase reporter gene assay for antibodies MOR-6032,MOR-7361, MOR-7480, and MOR-7483, all in IgG1 format.

TABLE 6 Characterization of IgG1 in Luciferase Reporter Gene AssayLuciferase reporter gene assay of IgGs Antibody plate-bound solublecross-linked MOR-6032 +++ − +++ MOR-7361 + − +++ MOR-7480 + − +++MOR-7483 + − +++

Example 4 Structural Characterization of Antibodies MOR-6032, MOR-7361,MOR-7480, and MOR-7483

The procedures described above in Examples 1-3 were used to produceseveral fully human anti-4-1BB IgG2 antibodies, including antibodiesdesignated as “MOR-6032”, “MOR-7361”, “MOR-7480”, and “MOR-7483.” ThecDNA sequences encoding the heavy and light chain variable regions ofthe MOR-6032, MOR-7361, MOR-7480, and MOR-7483 monoclonal antibodieswere obtained using standard PCR techniques and were sequenced usingstandard DNA sequencing techniques.

The nucleotide and amino acid sequences of the heavy chain variableregion, full length heavy chain of the IgG2 subclass, light chainvariable region, and full length light chain of antibodies MOR-6032,MOR-7361, MOR-7480, MOR-7480.1, MOR-7480.2, MOR-7483, MOR-7483.1, andMOR-7483.2 are provided in this disclosure; an index of the SEQ ID NOsfor these sequences is provided in Table 1.

Comparison of the MOR-6032 heavy chain immunoglobulin sequence to theknown human germline immunoglobulin heavy chain sequences demonstratedthat the MOR-6032 heavy chain utilizes a V_(H) segment from humangermline V_(H) 1-69, a D segment from the human germline 4-23, and a JHsegment from human germline JH 4a.

Further analysis of the MOR-6032 V_(H) sequence using the Kabat systemof CDR region determination led to the delineation of the H-CDR1, H-CDR2and H-CDR3 regions as shown in SEQ ID NOs: 1, 2 and 3, respectively.

Comparison of the MOR-7361 heavy chain immunoglobulin sequence to theknown human germline immunoglobulin heavy chain sequences demonstratedthat the 7361 heavy chain utilizes a V_(H) segment from human germlineV_(H) 3-23, a D segment from the human germline 2-8, and a JH segmentfrom human germline JH 4a.

Further analysis of the MOR-7361 V_(H) sequence using the Kabat systemof CDR region determination led to the delineation of the heavy chainH-CDR1, H-CDR2 and H-CDR3 regions as shown in SEQ ID NOs: 15, 16 and 17,respectively.

Comparison of the MOR-7480 and MOR-7483 heavy chain immunoglobulinsequences to the known human germline immunoglobulin heavy chainsequences demonstrated that the 7480 and 7483 heavy chains utilize aV_(H) segment from human germline V_(H) 5, a D segment from the humangermline 5-18, and a JH segment from human germline JH 4a.

Further analysis of the 7480 and 7483 V_(H) sequences using the Kabatsystem of CDR region determination led to the delineation of the H-CDR1,H-CDR2 and H-CDR3 regions as shown in SEQ ID NOs: 29, 30 and 31,respectively.

Comparison of the MOR-6032, MOR-7361, MOR-7480 and MOR-7483 light chainimmunoglobulin sequences to the known human germline immunoglobulinlight chain sequences demonstrated that the 6032, 7361, 7480 and 7483light chains all utilize a V_(L) segment from human germline A3-r and aJL segment from human germline JL 3b.

Further analysis of the MOR-6032 V_(L) sequence using the Kabat systemof CDR region determination led to the delineation of the light chainCDR1, CDR2 and CDR3 regions as shown in SEQ ID NOs: 6, 7, and 8,respectively.

Further analysis of the MOR-7361 V_(L) sequence using the Kabat systemof CDR region determination led to the delineation of the L-CDR1, L-CDR2and L-CDR3 regions as shown in SEQ ID NOs: 20, 21, and 22, respectively.

Further analysis of the MOR-7480 V_(L) sequence using the Kabat systemof CDR region determination led to the delineation of the L-CDR1, L-CDR2and L-CDR3 regions as shown in SEQ ID NOs: 34, 35, and 36, respectively.

Further analysis of the MOR-7483 V_(L) sequence using the Kabat systemof CDR region determination led to the delineation of the L-CDR1, L-CDR2and L-CDR3 regions as shown in SEQ ID NOs: 34, 35, and 55, respectively.

Example 5 Germlined Versions of Antibodies MOR-7480 and MOR-7483

In order to minimize immunogenicity of the MOR-7480 and MOR-7483antibodies, several amino acid residues were mutated back to germlinesequence, as follows. A germlined version of MOR-7480, designatedMOR-7480.1, was prepared by returning two amino acids in the FR1 regionof the heavy variable chain to germline sequence. More specifically, Qat amino acid residue number 1 was returned to the germline E, and K atamino acid residue number 19 was returned to R. The two amino acidresidues that were changed in the heavy chain variable region can beseen by comparing the amino acid sequence of MOR-7480 (SEQ ID NO:32)with MOR-7480.1 (SEQ ID NO:43). In the light chain variable region ofMOR-7480, five amino acids in the FR1 region (D1S, I2Y, A13S,R19S,S21T), two amino acids in the FR2 region (A42S, V45L), and one in theFR3 region (E80M) were reverted to germline sequence. The eight aminoacids that were changed in the light chain variable region can be seenby comparing the amino acid sequence of MOR-7480 (SEQ ID NO:37) withthat of MOR-7480.1 (SEQ ID NO:45).

Moreover, a third version of MOR-7480 was prepared by starting with thelight chain variable region sequence of MOR-7480.1 (SEQ ID NO:45) andreverting L45 back to V to produce MOR-7480.2 (SEQ ID NO:51).

A “germlined” version of MOR-7483, designated MOR-7483.1, was preparedby backmutating two amino acids in the FR1 region of the heavy variablechain to germline sequence. The germlined versions can be prepared bystarting with the germline version of the antibody chain and thenchanging the desired amino acids in the CDRs, or any combination ofmutations starting from any version. To produce MOR-7483.1, Q at aminoacid residue number 1 was returned to the germline E, and K at aminoacid residue number 19 was returned to R. The two amino acid residuesthat were changed in the heavy chain variable region can be seen bycomparing the sequence of MOR-7483 (SEQ ID NO:32) with MOR-7483.1 (SEQID NO:43). In the light chain variable region of MOR-7483, five aminoacids in the FR1 region (D1S, I2Y, A135, R195, S21T), two amino acids inthe FR2 region (A42S, V45L), and one in the FR3 region (E80M) werereverted to germline sequence. The eight amino acids that were changedin the light chain variable region can be seen by comparing the aminoacid sequence of MOR-7483 (SEQ ID NO:56) with that of MOR-7483.1 (SEQ IDNO:60).

Moreover, a third version of MOR-7483 was prepared by back mutating L45of the light chain variable region sequence of MOR-7483.1 (SEQ ID NO:60)to the germline V45 to produce MOR-7483.2 (SEQ ID NO:64).

Example 6 In Vitro Properties of Antibodies, Including GermlinedVersions

Binding Affinities of Antibodies (BIAcore Assay)

Binding kinetics of certain antibodies binding human 4-1BB were measuredby surface plasmon resonance (SPR) technology using a Biacore 3000instrument (GE Healthcare). Recombinant human 4-1BB/Fc Chimera proteincomprising amino acids 24-186 of SEQ ID NO: 68 was purchased from R&DSystems Inc. (#838-4B). The lyophilized protein was dissolved in abuffer containing 150 mM NaCl, 25 mM HEPES pH 8.0, 6 mM MgCl₂, 0.005%polysorbate 20, and 0.5 mM sodium azide to a final concentration of 80nM based on the predicted molecular weight (44.8 kDa) provided by theR&D Systems. The Fc portion of the molecule was cleaved by treatmentwith Bovine Factor Xa (Pierce, #32521) in 150 mM NaCl, 25 mM HEPES pH8.0, 6 mM MgCl₂, 0.005% polysorbate 20, 0.5 mM sodium azide, using a 20hour incubation at 22° C. with a 3% Factor Xa (3 μg Factor Xa per 100 μg4-1BB chimera). The 4-1BB portion of the molecule comprises amino acidresidues 24 though 186 of the human 4-1BB protein. Binding experimentswere carried out at 25° C. in a running buffer comprising 150 mM NaCl,25 mM HEPES pH 8.0, 6 mM MgCl₂, 0.005% polysorbate 20, and 0.5 mM sodiumazide. Antibodies were immobilized by standard amine coupling to a CM5sensorchip (GE Healthcare) using a 0.1 mg/mL solution of the antibody in10 mM sodium acetate at pH 5.0. The 4-1BB was injected at a range ofconcentrations from 80 nM to 0.16 nM, at a 50 μL/minute flow rate, for3.6 minutes followed by a 26 minute dissociation period using theKinject feature of the Biacore 3000 instrument. The bound complex wasregenerated by a 1 minute pulse of 10 mM phosphoric acid in water. Dataanalysis was performed using the Scrubber2 software (BioLogic Software).Sensograms were fit to a simple 1:1 Langmuir binding model. Theantibodies were shown to reversibly bind to recombinant human 4-1BB. Theresults (average values) are presented in Table 7.

Binding to the Extracellular Domain of 4-1BB (ELISA Assay)

Human 4-1BB IgG1Fc chimera (R&D Systems, Minneapolis, Minn.) wasresuspended with Dulbecco's Phosphate Buffered Saline (DPBS) containing0.1% bovine serum albumin (BSA) to 0.2 mg/ml and diluted with DPBS to afinal concentration of 0.03 ug/ml. Nunc-Immuno Maxisorp 96 well plateswere coated with 0.1 ml per well of the recombinant 4-1BB chimeraleaving empty wells for nonspecific binding controls and incubated at 4°C. overnight. The 4-1BB solution was removed and the plates were washedthree times with 0.2 ml wash buffer (0.05% Tween-20 in DPBS). 0.2 mlblocking buffer (5% BSA, 0.05% Tween-20 in DPBS) was added to all wellsand incubated at 4° C. for 1 hour with mixing. The blocking buffer wasremoved and plates washed three times with 0.2 ml wash buffer. Serialdilutions of the 4-1BB test antibodies were prepared in DPBS and 0.1 mldiluted Ab was added per well. Plates were incubated 1.5 hour at roomtemperature. Antibody solution was removed and the plates washed threetimes with 0.2 ml wash buffer per well. Horseradish peroxidase labeledgoat anti-human IgG, F(ab′)2 specific F(ab′)2 antibody (JacksonImmunoresearch #109-036-097, West Grove, Pa.) was diluted 1:5000 withDPBS and added 0.1 ml per well. The plates were incubated 1 hour at roomtemperature and washed with 0.2 ml per well wash buffer. 0.1 ml SureBlueTMB microwell peroxidase substrate (Kirkegaard & Perry Labs,Gaithersburg, Md.) was added and incubated for 20 minutes at roomtemperature. The reaction was stopped by adding an equal volume of 2MH₂SO₄ and absorbance was read at 450 nm on a Molecular Devices SpectraMax 340 (Molecular Devices, Sunnyvale, Calif.). The results arepresented in Table 8.

Ligand Competition Binding (ELISA Assay)

Antibodies were tested for their ability to block the binding of thehuman 4-1BB_IgG1Fc chimera to plate bound recombinant 4-1BB ligand(4-1BBL). Recombinant human 4-1BB ligand (Biosource/Invitrogen,Carlsbad, Calif.) was resuspended to 0.2 mg/mL in DPBS+0.1% bovine serumalbumin and then diluted to 1 μg/mL in DPBS. Nunc-Immuno MaxiSorpsurface 96 well plates were coated with 0.1 mL/well of the 4-1BBLsolution overnight at 4° C. The following day the 4-1BBL solution wasremoved and 0.2 mL Blocking buffer (1% bovine serum albumin, 0.05%Tween-20 in DPBS) added and incubated at room temperature for 2 hours.During the blocking step the antibody stocks were diluted in a rangefrom 8 ng/mL to 6 μg/mL in DPBS. Recombinant human 4-1BB_IgG1Fc (R&DSystems, Minneapolis, Minn.) was resuspended to 0.2 mg/mL in DPBS+0.1%bovine serum albumin and then diluted to 0.02 μg/mL in DPBS. The blocked4-1 BBL coated plates were washed three times with 0.2 mL wash buffer(0.05% Tween 20 in DPBS). 60 μL antibody dilutions were added along with60 μL 4-1BB_IgG1 Fc chimera and incubated at room temperature for 1.5hours. Plates were washed as described previously. Horseradishperoxidase anti-6× Histidine tag antibody (R&D Systems, MinneapolisMinn. #MAB050H) was diluted 1:1000 in DPBS, 50 μL of the resultingsolution added to the wells of the washed plates, and incubated at roomtemperature for 1 hour. Plates were washed as previously described, 50μL TMB substrate solution was added to each well and incubated at roomtemperature for 20 minutes. The reaction was stopped with 50 μL 0.2NH₂SO₄ and absorbance at 450 nm read using a Molecular Devices platereader. The results are presented in Table 8.

Species Cross-Reactivity of Antibodies

The species-cross reactivity of the exemplary antibodies was measuredusing phytohemagglutinin (PHA) stimulated primary peripheral bloodmononuclear cells (PBMC) of human, cynomolgus monkey (cyno), dog, andrat. Cells were isolated according to the procedure described below.Cells (˜5.0×10⁵ cells/tube) were washed once in cold wash buffer (PBS,2% FBS and 0.02% sodium azide) and 100 μl/tube of Alexa Fluor 647conjugated control or 4-1BB reactive antibodies at 15.5 μg/mL (100 nM)was added to each sample along with labeled species specific T cellmarker antibodies. The T cell marker antibodies utilized are as follows,FITC anti-human CD3e (BD Pharmingen, #555332), FITC anti-rat CD3e (BDPharmingen, #559975), FITC anti-rabbit CD4+ FITC anti-rabbit CD8 (AbDSerotec, #MCA799F and #MCA1576F), FITC anti-dog CD3e (AbD Serotec,#MCA1774F), and PerCP anti-human/cyno CD3e (BD Pharmingen, #552851). Thecells were incubated in the dark with fluorochrome antibodies on ice for30 minutes, washed three times and resuspended in 0.3 ml wash buffer foranalysis. Antibody staining was measured and analyzed using a BectonDickinson FACS Calibur and FlowJo 8.8.2 software.

Isolation of Human T Lymphocytes.

Human whole blood was collected into syringes containing 1 mL 0.5M EDTAand then transferred to Sigma Accuspin tubes (Sigma, St. Louis, Mo.) forthe isolation of peripheral blood mononuclear cells (PBMC) as describedby the manufacturer. The PBMCs were washed twice with DPBS containing 5mM EDTA and T lymphocytes were isolated using a T cell purificationcolumn as described by the manufacturer (R&D Systems, Minneapolis,Minn.). Briefly, PBMCs were resuspended in 2 mL of column buffer andloaded into a pre-washed T cell isolation column. PBMCs were incubatedfor 10 minutes at room temperature and T cells were eluted with columnbuffer, washed one time and resuspended TCM at 2×10⁶ cells/mL consistingof RPMI 1640 (Sigma, St Louis, Mo.) supplemented with 10% fetal bovineserum (Sigma, St. Louis, Mo.) and L-glutamine (2 mM), Hepes (10 mM),penicillin (100 U/ml), streptomycin (50 ug/ml) (Gibco, Grand Island,N.Y.).

Isolation of Cynomolgus PBMCs.

Cynomolgus whole blood (Bioreclamation; Hicksville, N.Y.) was collectedin sodium citrate CPT vacutainer tubes (BD; Franklin Lakes, N.J.) andthen spun at 1500×g for 20 minutes at room temperature. Tubes wereshipped overnight at 4° C. The PBMC fraction was collected from the CPTtubes and washed 2× with PBS containing 5 mM EDTA. Following the washstep, PBMCs were counted and readjusted to 2×10⁶ cells/mL in tissueculture medium (TCM). TCM consisted of RPMI 1640 (Sigma, St Louis, Mo.)supplemented with 10% fetal bovine serum (Sigma, St Louis, Mo.) andL-glutamine (2 mM), HEPES (10 mM), penicillin (100 U/mL), streptomycin(50 μg/mL) purchased from Gibco (Grand Island, N.Y.). Cells werestimulated with 10 μg/mL PHA 2-3 days to induce expression of 4-1BB.

Isolation of Canine PBMCs.

Canine whole blood was drawn into heparinized vacutainer tubes (BD;Franklin Lakes, N.J.) and diluted 1:2 with PBS containing 5 mM EDTA.After mixing, 4 mL of the diluted blood was carefully layered over 3 mLLympholyte-Mammal (Cedarlane Laboratories, Westbury, N.Y.) andcentrifuged 800×g for 20 minutes at 25° C. The PBMC interface wascollected, washed twice with PBS and resuspended to 2×10⁶ cells/mL inTCM containing PHA at 10 μg/mL (Remel, Lenexa, Kans.). The cells werecultured for 48-72 hours prior to testing for antibody binding by flowcytometry.

Isolation of Rat PBMCs.

Rat whole blood was drawn into heparinized vacutainer tubes (BD;Franklin Lakes, N.J.) and diluted 1:3 with PBS containing 5 mM EDTA.After mixing, 6 mL of the diluted blood was carefully layered over 4.5ml Lympholyte-Mammal (Cedarlane Laboratories, Westbury, N.Y.) andcentrifuged 800×g for 20 minutes at 25° C. The PBMC interface wascollected, washed twice with PBS and resuspended to 2×10⁶ cells/mL inTCM containing PHA at 10 μg/mL (Remel, Lenexa, Kans.). The cells werecultured for 48-72 hours prior to testing for antibody binding by flowcytometry.

The binding results are provided in FIG. 1. The antibodies were found tobind to the human and cyno 4-1BB with high affinity, while binding todog and rat 4-1BB was not observed at concentrations of 100 nM, thehighest concentration tested.

TABLE 7 Binding Affinities of IgG Antibodies (Biacore) Antibody k_(a)(M⁻¹ s⁻¹) k_(d) (s⁻¹) K_(D) (nM) t_(1/2) MOR7480 3.6 × 10⁵ 1.5 × 10⁻⁴0.42  82 min (IgG1) MOR7480 4.5 × 10⁵ 2.3 × 10⁻⁴ 0.57  50 min (IgG2)MOR-7480.1 1.2 ± 0.3 × 10⁶ 9.8 ± 8.4 ± 1.4 1.2 min (IgG1) 0.15 × 10⁻³ MOR-7480.1 1.4 ± 0.06 × 1.2 ± 0.1 × 10⁻² 8.7 ± 1.0 1.0 min (IgG2) 10⁶MOR-7480.2 9.3 × 10⁵ 4.1 × 10⁻⁴ 0.4  28 min (IgG1) MOR-7483 6.0 × 10⁵4.4 × 10⁻⁴ 0.73  26 min (IgG1) MOR-7483 3.0 × 10⁵ 3.8 × 10⁻⁴ 1.3 1.3 min(IgG2) MOR-7483.1 8.0 × 10⁵ 0.022 28  32 min (IgG1)

TABLE 8 ELISA Binding and Ligand Competition Values Binding ELISA LigandCompetition ELISA EC50 ± STD % max IC50 ± STD Antibody Isotype (nM)inhibition (nM) MOR-6032 IgG1 0.071 ± 0.029 100 ± 2 0.153 ± 0.067MOR-6032 IgG4 0.226 ± 0.161 100 ± 2 0.112 ± 0.023 MOR-7361 IgG1 0.091 ±0.010 100 ± 2 0.172 ± 0.006 MOR-7480 IgG1 0.076 ± 0.008  96 ± 2 0.122 ±0.019 MOR-7480 IgG2 0.122 ± 0.009  98 ± 2 0.125 ± 0.003 MOR-7483 IgG10.073 ± 0.024  98 ± 2 0.109 ± 0.028 MOR-7483 IgG2 0.165 ± 0.035  97 ± 20.138 ± 0.015

Epitope Mapping

In order to determine the epitope binding region of the 4-1BB agonistantibodies, a series of mutations (Table 9) were made to the human 4-1BBextracellular domain to the published dog 4-1BB sequence (Ref. Seq.XM_(—)845243).

TABLE 9 Mutant of Human 4-1BB Extracellular Domain Mutant of human 4-1BBextracellular domain Amino Acid Changes Hu41BB N&E N30K, A56T, G57S,R60K, T61A Hu41BB N30K, D38G, N39K, R41K, S46I, A56T, G57S, R60K, N&E.1T61A Hu41BB N30K, A56T, G57S, R60K, T61A, K69E, R75K, E77V N&E.2 Hu41BBL24I, P27S, N42S, T89I, P90S, S100T N&E.3 Hu41BB L24I, P27S, N30K, D38G,N39K, R41K, N42S, S46I, N&E.4 A56T, G57S, R60K, T61A, K69E, R75K, E77V,T89I, P90S, S100T Hu41BB K115Q, C121R, R134Q, R154S, V156A N&E.5 Hu41BBS161A, P162S, D164G, L165F, A169T N&E.6

All human-to-dog mutations were prepared by Gene Dynamics LLC,(Portland, Oreg.) in the retroviral expression vector pMSCVpuro(Clontech Laboratories Mountain View, Calif.). Additionally the fullcanine cDNA sequence was prepared via gene synthesis corresponding toRef. Seq. XM_(—)845243.

Viral preparations were established by transient transfection of roughly40-50% confluent 293T cells in T-75 flasks. Following culture, the viralsupernatant was then sterile filtered, and subjected to concentration.The concentrated virus was collected and stored at −80° C.

Logarithmically growing 300-19 cells were transduced with retrovirususing 1:250 dilution concentrated virus plus 8 ug/ml polybrene incomplete DMEM. Following a 24-hour incubation, 2 ug/ml puromycin wasadded to the cultures and maintained during the course of the study.

Positive expression of the 4-1BB receptors by the puromycin selectedpools was confirmed by staining with 1 ug/ml polyclonal goat anti-human4-1BB antibody (R&D Systems Inc.) plus 1:200 dilution PE labeled donkeyanti-goat IgG (H+L) F(ab′)₂ (Jackson Immunoresearch Inc.). In order todetermine recognition of the mutant 4-1BB receptors by the testantibodies the puromycin selected pools were stained with 100 nMdilution of the unlabeled primary antibody on ice for 30 min, followedby two washes with FACS buffer, and 1:200 dilution species specific PElabeled donkey anti-IgG (H+L) F(ab′)₂. Cells were analyzed by FACS usinga BD FACS Calibur and FlowJo 8.8.6 software.

Relative staining of each cell pool is summarized in Table 10.

TABLE 10 Relative Staining of Each Cell Pool Dog Ab h41BB N&E N&E.1N&E.2 N&E.3 N&E.4 N&E.5 N&E.6 41BB Goat pAb + + + + + + + + + BBK-2 + +− + + − + + − JG1.6A + + − + + − + + − 4B4-1 + + − + + − + + −6032_G1 + + + − + − + + − 7361_G1 + + + − +/− − + + −7480_G1 + + + + + + + + + 7480.1_G1 + + + + + + − + −7480.1_G2 + + + + + + +/− + − 7480.2_G1 + + + + + + + + +7483_G1 + + + + + + +/− + +/− 7483_G2 + + + + + + − + −

Differentiation of binding between antibodies having similar sequences(MOR-7480, MOR-7480.1, MOR-7480.2, MOR-7483, and MOR-7483.1) wasdiscovered within the mutations of clone N&E.5, suggesting that thedeterminants for antibody recognition lie within the mutated region.

In order to determine the relative affinity of these antibodies forhuman 4-1BB extracellular domain and the mutant of human 4-1BBextracellular domain, mutant N&E.5, a dose response FACS curve wasdetermined for each antibody. Alexa Fluor 647 labeled MOR 7480, MOR7480.1, and MOR 7480.2 were diluted in FACS buffer from 1 uM in an 8point 1:5 dilution series and used to stain parental 300-19, hu4-1BB,hu4-1BB N&E.5, and dog 4-1BB cell pools. The cells were analyzed by FACSusing a BD FACS Calibur and FlowJo 8.8.6 software. The geometric meanfluorescence of each receptor expressing pool was normalized to stainingof parental cells and expressed as fold staining and an EC50 for doseresponse was determined. The EC50 summary is shown in Table 11. Greaterthan 5 fold decrease in binding for both MOR_(—)7480.2 and MOR_(—)7480for the human 4-1BB mutant N&E.5 was noted.

TABLE 11 Binding EC₅₀ (nM) of Antibodies 4-1BB Mutant Antibody (IgG1)Human 4-1BB N&E.5 MOR_7480.1 7.916 n/a MOR_7480.2 0.510 2.730 MOR_74801.68 12.29

Agonist Activity of Antibodies (Luciferase Activity Assay)

293T cells expressing human 4-1BB along with a stably integrated NFkBluciferase reporter were prepared. Cells were harvested, washed andresuspended into phenol red free complete medium (DMEM containing 10%fetal bovine serum, HEPES buffer, nonessential amino acids andL-glutamine) at density of 0.6×10⁶ cells/mL. 50 μl of cells were platedinto each assay well of a white 96 well plate (PerkinElmer, Waltham,Mass.). Test antibodies were added to each well in the presence 2.5:1ratio of a cross linking antibody Fab′ goat anti-human IgG Fc (JacksonImmunoResearch, West Grove, Pa.). The plate was incubated 5 hours at 37°C. 75 μl of Bright-Glo Luciferase reagent (Promega, Madison Wis.) wasadded and the amount of luciferase activity was measured using a PackardTopCount NXT scintillation counter.

293T cells expressing cynomologous monkey 4-1BB were prepared by viraltransduction and selection of a stable pool with 2 μg/ml puromycin. Cyno4-1BB expressing 293T cells were plated in a T-75 flask to roughly60-70% confluency then transfected with 10 μg pLuc_(—)6xNFkB plus 0.1 μgpRL-CMV as a transfection control. Transfections were performed usingFugene 6 transfection reagent (Roche Indianapolis, Ind.) at a 6 μlFugene to 1 μg plasmid DNA ratio according to manufacturer instructions.Cells were harvested the following day washed and resuspended intophenol red free complete medium (DMEM containing 10% fetal bovine serum,nonessential amino acids and L-glutamine) at density of 0.6×10⁶cells/mL. 50 μl of cells were plated into each assay well of a white 96well plate (PerkinElmer, Waltham, Mass.). Test antibodies were added toeach well in the presence 2.5:1 ratio of a cross linking Fab′ goatanti-human IgG Fc antibody (Jackson ImmunoResearch, West Grove, Pa.).The plate was incubated hours at 37° C. 75 μl of luciferase assayreagent was added and the amount of firefly luciferase activity wasmeasured using a Packard TopCount NXT scintillation counter.Additionally, 75 μl of Stop & Glo reagent was added to assess therenilla luciferase activity. The amount of renilla luciferase activitywas measured using a Pakcard TopCount NXT scintillation counter. Resultsare presented in FIG. 2.

Agonist Activity of Antibodies (Primary T Cell IL-2 Release Assay)

Nunc Maxisorp 96 well plates were UV sterilized prior to plate coating.Test antibodies were diluted in PBS to 60 μg/ml. 0.2 ml of the dilutedAb was divided to 2 wells of a polypropylene 96 well plate and seriallydiluted 1:3. 50 μl of the diluted Ab were added to the sterile Maxisorp96 well assay plate and immediately 50 μl of 20 μg/ml anti-human CD3cclone UCHT1 was added (Biolegend San Diego, Calif.). All plates werethen incubated overnight at 4° C. The following day, Ab coated plateswere washed 1× with PBS and 0.15 ml RPMI complete media was added to thewells of the Nunc Maxisorp plates. Human T cells were isolated asdescribed previously elsewhere herein and 50 μl purified T cells at2×10⁶ cells/ml (100,000 cells/well) were added to each well. Cells wereincubated at 37° C., 5% CO₂ for 3 days. Supernatant from each well wascollected and either assayed immediately or stored at −20° C. prior toassay. Supernatants were diluted with complete media prior to IL-2 ELISAassay (R&D Systems, Minneapolis, Minn.). Results are presented in FIG.3.

Example 7 Human Leukocyte Expansion Induced by 4-1BB Antibodies In Vivo

The lack of detectable cross-reactivity of the 4-1BB antibodies with themurine 4-1BB and the requirement for the presence of human immune cellsrequired the development of models for the in vivo functional assessmentof the 4-1BB antibodies. Mice with the NOD genetic background carryingthe severe combined immunodeficient (SCID) mutation and deficiency inthe IL-2 receptor common gamma chain (commonly termed NSG) are able tosupport the engraftment of large number of human peripheral bloodleukocytes (huPBL) and maintain engraftment for at least 30 days (King,2008, Clin. Immunol. 126:303-314). This mouse model, also known ashuPBL-NSG model, was used to assess the functional effect of in vivosystemic administration of the antibodies on human immune cells.Specifically, 6 million freshly isolated human PBMCs were adoptivelytransferred via intraveinous injection into NOD.Cg-Prkdc^(scid)II2rg^(tmlWjl)/SzJ (NSG) host mice. Nine days post PBMC injections, theanimals were administered a single 1 mg/kg dose of MOR7480, MOR7480.1 orIgG2 isotype control antibody via intraperitoneal injection. At day 24to 28 post PBMC engraftment, PBMC were stained with antibodies to humanand murine CD45 assessed via flow cytometry. Forward and side scatterprofiles were used to determine a lymphocyte gate. As shown in FIG. 4,MOR7480 and MOR7480.1 were able to enhance expansion of human leukocytesas evidenced by increased proportion of human CD45+ cells in theperipheral blood of engrafted mice. For each group, n≧6 mice.

In addition, MOR7480.1 treatment of cynomolgus monkeys increasedproliferation among cytotoxic central memory T cells (CD8 T_(CM)) inPBMC samples. Cynomolgus monkeys (2 animals per dose level) were given asingle intravenous injection of MOR7480.1 at the indicated dose. PBMCwere harvested 7 days prior to the antibody dose (pre dose) and on theindicated study days relative to administration of MOR7480.1 (on StudyDay 1). PBMC were stained with antibodies for CD3, CD4, CD8, CD28, CD95,and Ki-67 and analyzed via flow cytometry. Data was collected on a CantoII (Beckton Dickinson) and analyzed using DIVA software (BectonDickinson). CD8 central memory cells were identified as CD3+, CD8+,CD28+ and CD95+. Data is shown for individual animals designated as(dose level-animal number) and is represented as intra-animal change inthe number of Ki-67+ cells relative to pre study number {[(#Ki-67+ cellson indicated study day−#Ki-67+ cells at pre dose)/#Ki-67+ cells at predose]*100}. As shown in FIG. 5, A 1.5 fold or greater increase inproliferating central memory T cells during the first 7-13 days of studywas noted in at least one animal of all groups treated with 0.3 mg/kg orgreater.

Example 8 Anti-Tumor Activity of 4-1BB Antibodies (In Vivo Model)

PC3 Human Prostate Cancer Model.

The lack of rodent cross reactivity of the 4-1BB antibodies preventedthe use of standard murine syngeneic or human xenograft tumor models forthe assessment of anti-human tumor efficacy of the antibodies.Accordingly, a novel huPBL-SCID-Bg xenogenic tumor mouse model wasgenerated using a SCID-Bg mouse (CB.17/Icr.CgPkrdc^(scid)Lyst^(bg)/Crl), which harbors the beige (Bg) mutation lackmurine T and B lymphocytes and functional NK cells. The anti-human tumorefficacy of the 4-1BB antibodies was assessed using this model asdescribed below.

The PC3 human prostate or LOVO human colon cell line was obtained fromAmerican Type Culture Collection and was cultured in RPMI-1640(Invitrogen) enriched with the following Invitrogen supplements:L-Glutamine, Sodium pyruvate, non-essential amino acids,penicillin/streptomycin, Hepes, and 10% heat inactivated fetal bovineserum (FBS; Cat. No. F2442, Sigma Aldrich). Cells were grown toconfluency in T-225 Falcon flasks. Subsequently, cells were trypsinized(Trypsin 0.25%-EDTA; Cat. No. 2500-056, Invitrogen) and growth wasscaled up in Hyperflasks (Cat. No. 3319 Corning Life Sciences) for threedays. Trypsin was used to harvest the cell line which was washed 3 timesin ice cold PRMI supplemented with 10% FBS. No greater than 300 ml ofperipheral blood was collected from healthy volunteers. Peripheral bloodlymphocytes (PBMCs) were isolated from heparinized blood using Accuspintubes in accordance with the manufactures' protocol (Cat. No.A0561-100×15 ml, Aldrich). Counted cell suspensions were combined suchthat each mouse received an injection of 1.5×10⁶ PBMCs and 3×10⁶ tumorcells in a single bolus injection of 0.2 mL in PBS. The combined cellsuspension was washed twice with cold PBS, placed on ice and immediatelyinjected into prepared mice.

For each mouse, a 0.2 mL volume of the combined cell suspension wasinjected subcutaneously into the right flank of the animal and given asingle dose (0.2 mL) of the 4-1BB antibody or control antibody bysubcutaneous injection into the left flank. Tumor measurements were madevia Pressier caliper twice per week for the duration of the experimentsand body weights were also recorded. Tumor volume was calculated usingthe following calculation: length×width²×0.44=volume (mm³) Mice wereremoved from the study in the event that the tumor volume reached 2000mm³ or an animal lost 20% of body weight before termination of theexperiment. On day 23 mice from all groups were euthanized in accordancewith procedures outlined by the IACUC (FIG. 6). Percent tumor growthinhibition was measured on the final day of the study and is calculatedas 100−{1−(Treated_(final day)/Control_(final day))}. Similar resultswere observed when tumors were measured on day 6 post injection, and theanimals were randomized according to tumor volume, and given a single4-1BB mAb dose on day 7 post implantation. For most studies, each groupcontained 8 mice.

Example 9 In Vivo Assessment of Activity of 4-1BB Antibodies in Human4-1BB Knock in Mice

Generation of Human 4-1BB Knock-in Mice

To better address the immune modulating activities of anti-human 4-1BBmonoclonal antibodies that do not cross react with murine 4-1BB, a mousemodel in which the mouse 4-1BB gene was replaced by the human 4-1BB genewas generated. Bacterial artificial chromosome (BAC) clones carrying thehuman or murine 4-1BB genomic fragment were ordered from Invitrogen(Carlsbad, Calif.) and used to construct the 4-1BB targeting vectorbased on the Red/ET recombination technology (Zhang, 1998, Nat Genet.20:123-128). First, a retrieval vector was assembled on the pBR322backbone such that, when opened by Xba1 digestion, the two murine/humanchimeric homology arms (400 bps each) will retrieve from the human 4-1BBBAC clone the 19,994 bps of human 4-1BB genomic sequence beginning withthe translation start codon ATG located in exon 2 and ending with thestop codon TGA in exon 8. Second, a neomycin expression cassette underthe control of the PGK/EM7 promoters was assembled and flanked by 100base pairs (bps) of sequences homologous to intron 2 sequences of thehuman 4-1BB gene. This neomycin expression cassette was then targetedinto the retrieved human 4-1BB genomic fragment obtained in step 1.Lastly, the retrieved human 4-1BB genomic fragment carrying the neomycinexpression cassette was targeted into a murine BAC clone to replace themurine 4-1BB gene with the modified human 4-1BB genomic fragment fromATG start codon to TGA stop codon.

This BAC targeting vector was electroporated into a mouse embryonic stemcell line on the C57BL/6NTAC background (PRX-BL6N #1, Primogenix,Laurie, Mo.) following a standard protocol and clones surviving G418(also known as geneticin) selection were screened by two Taqman assaysagainst intron 2 and exon 8 of the murine 4-1BB gene to identify clonesthat had been modified at the murine 4-1BB locus by a homologousrecombination mechanism Of the 116 ES clones screened, 7 clones werefound to have lost one allele of the murine 4-1BB locus (targetingefficiency 6%). Karyotype analysis and in situ hybridization (FISH) wereperformed by Coriell Institute for Medical Research (Camden, N.J.). Forclone LH15, 19 out of 20 cells were 40 XY, and for LH80, 20 out of 20cells 40XY. In both clones, FISH using a murine BAC clone carrying the4-1BB gene as a probe showed one signal of 4-1BB hybridization on eachof the chromosome 4 pair in the region of band E2. No signal was seen atany other locations.

Both clones LH15 and LH80 were injected into blastocysts of the BALB/cstrain and embryos implanted into the CD1 pseudopregnant female mice tocarry to term. Male chimeras were mated to the Ella-cre mice on C57BL/6background to remove the neomycin resistance cassette and micehomozygous for the human 4-1BB gene were used in study.

4-1BB Agonist mAb Mediated Lymphocyte Proliferation.

The ability of 4-1BB agonist mAbs to induce lymphocyte proliferation wasassessed in 4-1BB Knock-in mice. 4-1BB Knock-in mice were dosed viaintraperitoneal injection with 30 mg/kg of MOR7480.1 on study Day 0 (forweekly dosing animals received 4-1BB mAb injections on Day 0 and Day7).24 hours prior to sample collection, animals were injectedintraperitoneally with 2 mg BrdU. At the indicated day post dose,peripheral blood samples were collected via intracardiac puncture. PBMCwere stained with antibodies against CD3, CD4, CD8, and BrdU andassessed via flow cytometry. Results are presented in FIG. 7 panel A.

4-1BB Agonist mAb Mediated Anti-Tumor Efficacy

Anti-tumor efficacy of MOR7480.1 was assessed in 4-1BB knock in miceusing B16-OVA/luc, a melanoma line that has been engineered to expressthe ovalbumin (OVA) model antigen and luciferase (luc). One milliontumor cells were implanted on the flank of 4-1BB knock in mice. Animalswere randomized based on tumor size on when tumors reached approximately50-100 mm³ (generally 7-10 days post tumor inoculation) and given asingle injection of the indicated dose of 4-1BB mAb. Tumor size wasassessed using caliper measurement two to three times per week untilstudy termination. Results are presented in FIG. 7 panel B.

The invention claimed is:
 1. An isolated antibody or an antigen-bindingportion thereof wherein said antibody or antigen-binding portion bindshuman 4-1BB extracellular domain at an epitope located within amino acidresidues 115-156 of SEQ ID NO:
 68. 2. The antibody or antigen-bindingportion thereof according to claim 1 wherein said antibody orantigen-binding portion binds human 4-1BB with a K_(D) of 10 nM or lessfor the human 4-1BB extracellular domain as measured using a surfaceplasmon resonance assay.
 3. The antibody or antigen-binding portionthereof according to claim 2 wherein said antibody or antigen-bindingportion thereof comprises a V_(H) and a V_(L) region wherein the V_(H)region amino acid sequence is set forth in SEQ ID NO: 43 and the V_(L)region amino acid sequence is set forth in SEQ ID NO:
 45. 4. Apharmaceutical composition comprising an antibody or antigen-bindingportion thereof according to claim 1 and a pharmaceutically acceptablecarrier.
 5. A method for reducing tumor growth in a subject in needthereof, comprising administering to the subject an effective amount ofan antibody or antigen-binding portion thereof according to claim 1, anda pharmaceutically acceptable carrier.
 6. A method of treating cancer ina mammal, which comprises administering to the mammal in need oftreatment a therapeutically effective amount of an antibody orantigen-binding portion thereof according to claim 1, and apharmaceutically acceptable carrier.
 7. The method of claim 6 whereinsaid cancer is selected from the group consisting of colorectal cancer,non-Hodgkin's lymphoma, prostate cancer, or melanoma.
 8. A method oftreating cancer in a mammal which comprises administering to the mammala therapeutically effective amount of an antibody or antigen-bindingportion thereof according to claim 3 in combination with animmunotherapeutic agent.
 9. The method of claim 8 wherein saidimmunotherapeutic agent is rituximab.
 10. A method for reducing tumorgrowth in a subject in need thereof, comprising administering to thesubject an effective amount of an antibody or antigen-binding portionthereof according to claim 3, and a pharmaceutically acceptable carrier.11. A method of treating cancer in a mammal, which comprisesadministering to the mammal in need of treatment a therapeuticallyeffective amount of an antibody or antigen-binding portion thereofaccording to claim 3, and a pharmaceutically acceptable carrier.
 12. Themethod of claim 11 wherein said cancer is selected from the groupconsisting of colorectal cancer, non-Hodgkin's lymphoma, prostatecancer, or melanoma.
 13. A method of treating cancer in a mammal whichcomprises administering to the mammal a therapeutically effective amountof an antibody or antigen-binding portion thereof according to claim 1in combination with an immunotherapeutic agent.
 14. The method of claim13 wherein said immunotherapeutic agent is rituximab.